Insulin derivative

ABSTRACT

The present invention relates to novel human insulin derivatives which are soluble at physiological pH values and have a prolonged profile of action. The invention also relates to methods of providing such derivatives, to pharmaceutical compositions containing them, to methods of treating diabetes and hyperglycaemia using the insulin derivatives of the invention and to the use of such insulin derivatives in the treatment of diabetes and hyperglycaemia.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. §371 national stage application of International Patent Application PCT/EP2007/054444 (published as WO 2007/128817 A2), filed May 8, 2007, which claimed priority of European Patent Application 06113709.7, filed May 9, 2006 and European Patent Application 06118260.6, filed Aug. 1, 2006; this application further claims priority under 35 U.S.C. §119 of U.S. Provisional Application 60/801,122, filed May 16, 2006 and U.S. Provisional Application 60/835,354, filed Aug. 3, 2006.

FIELD OF THE INVENTION

The present invention relates to novel human insulin derivatives which are soluble at physiological pH values and have a prolonged profile of action. The invention also relates to methods of providing such derivatives, to pharmaceutical compositions containing them, to methods of treating diabetes and hyperglycaemia using the insulin derivatives of the invention and to the use of such insulin derivatives in the treatment of diabetes and hyperglycaemia.

BACKGROUND OF THE INVENTION

Currently, the treatment of diabetes, both type 1 diabetes and type 2 diabetes, relies to an increasing extent on the so-called intensive insulin treatment. According to this regimen, the patients are treated with multiple daily insulin injections comprising one or two daily injections of a long acting insulin to cover the basal insulin requirement supplemented by bolus injections of a rapid acting insulin to cover the insulin requirement related to meals.

Long acting insulin compositions are well known in the art. Thus, one main type of long acting insulin compositions comprises injectable aqueous suspensions of insulin crystals or amorphous insulin. In these compositions, the insulin compounds utilized typically are protamine insulin, zinc insulin or protamine zinc insulin.

Certain drawbacks are associated with the use of insulin suspensions. Thus, in order to secure an accurate dosing, the insulin particles must be suspended homogeneously by gentle shaking before a defined volume of the suspension is withdrawn from a vial or expelled from a cartridge. Also, for the storage of insulin suspensions, the temperature must be kept within more narrow limits than for insulin solutions in order to avoid lump formation or coagulation.

While it was earlier believed that protamines were non-immunogenic, it has now turned out that protamines can be immunogenic in man and that their use for medical purposes may lead to formation of antibodies. Also, evidence has been found that the protamine-insulin complex is itself immunogenic. Therefore, with some patients the use of long acting insulin compositions containing protamines must be avoided.

Another type of long acting insulin compositions are solutions having a pH value below physiological pH from which the insulin will precipitate because of the rise in the pH value when the solution is injected. A drawback with these solutions is that the particle size distribution of the precipitate formed in the tissue on injection, and thus the release profile of the medication, depends on the blood flow at the injection site and other parameters in a somewhat unpredictable manner. A further drawback is that the solid particles of the insulin may act as a local irritant causing inflammation of the tissue at the site of injection.

International patent application published under number WO 2005/012347 (Novo Nordisk A/S) concerns insulin derivatives which have a sidechain attached to either the α-amino group of the N-terminal amino acid residue of the B-chain or the ε-amino group of a Lys residue present in the B chain.

International patent application No. EP2006/050593 (Novo Nordisk A/S) discloses insulin derivatives having an aromatic group in the side chain.

Patent application no. EP2006/050594 (Novo Nordisk A/S), disclose insulin derivatives having a PEG in the side chain.

Other insulin derivatives are disclosed in JP laid-open patent application No. 1-254699 (Kodama Co., Ltd.) and WO 95/07931 (Novo Nordisk A/S).

However, there is still a need for insulin having a more prolonged profile of action than the insulin derivatives known up till now.

SUMMARY OF THE INVENTION

In one aspect of the invention an insulin derivative is having a formula

wherein Ins is a parent insulin moiety and Q₁-Q₂-[CH₂]_(n)—X₁—[CH₂]_(n)-Q₃-[CH₂]_(n)—X₂—[CH₂]_(n)-Q₄-[CH₂]_(n)—X₃—[CH₂]_(n)-Q₅-[CH₂]_(n)—Z is a substituent and where the Ins is attached to the substituent via an amide bond between the α-amino group of the N-terminal amino acid residue of the B chain of Ins or an ε-amino group of a Lys residue present in the A or B chain of Ins and a CO group in Q₁ or Q₂ of the substituent; each n is independently 0, 1, 2, 3, 4, 5 or 6; Q₁ is:

-   -   an amino acid amide of an amino acid with a carboxylic acid in         the side chain, or an amino acid with an uncharged side chain,         which residue forms, with its carboxylic acid group, an amide         group together with the α-amino group of the N-terminal amino         acid residue of the B chain of Ins or together with the ε-amino         group of a Lys residue present in the A or B chain of Ins, or

a chain composed of two, three or four α-amino acid amide or amino acid residues as specified above linked together via amide bonds, which chain—via an amide bond—is linked to the α-amino group of the N-terminal amino acid residue of the B chain of Ins or to the ε-amino group of a Lys residue present in the A or B chain of Ins, or

a bond

Q₂ is:

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—

—CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—;

—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃; or

a bond

-   -   provided that         -   at least one of Q₁ or Q₂ is not a bond, and         -   that Q₂ is not —CO—(CH₂)₂—CO—NH— when n is 0 or 1, X₁ is a             bond and Q₃ is (CH₂CH₂O)₂—, (CH₂CH₂O)₃— or             (CH₂CH₂OCH₂CH₂CH₂CH₂O)— and         -   that if an amine in Q₁ or Q₂ forms a bond with the rest of             the substituent, the amine must be bound to the rest of the             substituent via a carbonyl group;             Q₃, Q₄, and Q₅ independently of each other can be

—(CH₂)_(m)— where m is an integer in the range of 6 to 32;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH₂— groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 32;

—CO—((CR⁵R⁶)₁₋₆—NH—CO)—;

—(CO—(CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃;

—CO—(CH₂)₀₋₃—Ar—(CH₂)₀₋₃— where Ar can be arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R² where R¹ and R² independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃;

(CH₂CH₂O)_(y)—; (CH₂CH₂CH₂O)_(y)—; (CH₂CH₂CH₂CH₂O)_(y)—; (CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)— or (CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)—; —(CH₂OCH₂)_(y)— where y is 1-20;

arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R² where R¹ and R², independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃;

a chain of the formula —(CH₂)_(s)—Y₁—(Ar)_(v1)—Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)— wherein Ar is defined as above, Y₁-Y₆ independently of each other can be O, S, S═O, SO₂ or a bond; where s, w, t and z independently of each other are zero or an integer from 1 to 10 so that the sum of s, w, t and z is in the range from 4 to 30, and v₁, v₂, and v₃ independently of each other can be zero or 1 with the proviso that Y₁-Y₆ do not link to each other and that the structure —O—(CH₂)₁—O— does not occur; or

a bond;

with the proviso that at least one of Q₃-Q₅ is not a bond;

X₁, X₂ and X₃ are independently of each other

O;

—C═O

a bond;

NCOR¹, where R¹ can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;

with the proviso that X₁, X₂ and X₃ cannot bind to Z and when X₁, X₂ and X₃ are O, then X₁, X₂ and X₃ do not bind directly to O in Q₃, Q₄, and Q₅

and

Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)₂;

—N(CH₂COOH)₂;

—SO₃H

—OSO₃H

—OPO3H₂

—PO₃H₂ or

-tetrazol-5-yl or

—O—W₁,

where W₁ is arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of tetrazo-5-lyl, —COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONR³R⁴ or —SO₂NR³R⁴ where R³ and R⁴, independently of each other can be H, —(CH₂)₁₋₆—SO₃H, or —(CH₂)₁₋₆—O—PO₃H₂; provided that when Z is —O—W₁ then Q₁ must be present and any Zn²⁺ complex thereof

In one aspect of the invention Q₂ of the insulin derivative is selected from the group consisting of

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—;

—CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—; or

—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃

In one aspect of the invention Q₃ is —(CH₂)_(m)— where m is an integer in the range of 6 to 32 or from 8 to 20 or m is 12, 13, 14, 15 or 16.

In one aspect of the invention Q₁, Q₄, Q₅, X₁, X₂ and X₃ is bonds and n is zero.

In one aspect of the invention Q₃ is —(CH₂)_(m)— where m is an integer in the range of 6 to 32 or from 8 to 20 or m is 12, 13, 14, 15 or 16, Q₁, Q₄, Q₅, X₁, X₂ and X₃ is bonds, n is zero and Z is —COOH.

In one aspect of the invention one of Q₃, Q₄, or Q₅ is (CH₂CH₂O)_(y)—; (CH₂CH₂CH₂O)_(y)—; (CH₂CH₂CH₂CH₂O)_(y)—; (CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)— or (CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)—; —(CH₂OCH₂)_(y)— where y is 1-20.

In one aspect of the invention Q₃ is

—CO—((CR⁵R⁶)₁₋₆—NH—CO)—;

—(CO—(CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃;

—CO—(CH₂)₀₋₃—Ar—(CH₂)₀₋₃— where Ar can be arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R² where R¹ and R² independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

a bond

Q₄ is

—(CH₂)_(m)— where m is an integer from 4 to 22;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH₂— groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 22;

arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R² where R¹ and R², independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

-   -   a chain of the formula         —(CH₂)_(s)—Y₁—(Ar)_(v1)—Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)—         wherein Ar is defined as above, Y₁-Y₆ independently of each         other can be O, S, S═O, SO₂ or a bond; where s, w, t and z         independently of each other are zero or an integer from 1 to 10         so that the sum of s, w, t and z is in the range from 4 to 30,         and v₁, v₂, and v₃ independently of each other can be zero or 1         with the proviso that Y₁-Y₆ do not link to each other and that         the structure —O—(CH₂)₁—O— does not occur;

X₁ is

O;

—C═O

NCOR¹, where R¹ can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

-   -   where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;     -   with the proviso that when X₁ is O then X₁ does not bind         directly to O in Q₄;         X₂, X₃ and Q₅ are bonds;         All values of n are zero; and         Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)₂;

—N(CH₂COOH)₂;

—SO₃H

—OSO₃H

—OPO3H₂

—PO₃H₂ or

-tetrazol-5-yl or

—O—W₁,

where W₁ is arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of tetrazo-5-lyl, —COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONR³R⁴ or —SO₂NR³R⁴ where R³ and R⁴ independently of each other can be H, —(CH₂)₁₋₆—SO₃H, or —(CH₂)₁₋₆—O—PO₃H₂; and any Zn²⁺ complex thereof.

In one aspect of the invention Z is —COOH.

In one aspect of the invention the parent insulin of the insulin derivative is an insulin analogue.

In one aspect of the invention the parent insulin is selected from the group consisting of: desB30 human insulin, GlyA21 human insulin, GlyA21desB30 human insulin, GlyA21ArgB31ArgB32 human insulin, LysB3GluB29 human insulin, LysB28ProB29 human insulin and ThrB29LysB30 human insulin.

In one aspect of the invention there is provided a pharmaceutical composition for the treatment of diabetes in a patient in need of such treatment, comprising a therapeutically effective amount of an insulin derivative according to the invention.

In one aspect of the invention there is provided a method for producing a pharmaceutical composition comprising a therapeutically effective amount of an insulin derivative according to the invention, wherein up to about 10 zinc atoms per 6 molecules of insulin derivative are added to the pharmaceutical composition.

In one aspect of the invention there is provided a method of treating diabetes in a patient in need of such a treatment, comprising administering to the patient a therapeutically effective amount of an insulin derivative according to the invention.

In one aspect of the invention the insulin derivative is administered pulmonary.

In one aspect of the invention there is provided a mixture of an insulin derivative according to the invention and a rapid acting insulin analogue selected from the group consisting of AspB28 human insulin; LysB28ProB29 human insulin and LysB3GluB29 human insulin.

In one aspect of the invention the insulin derivative is selected from the group consisting of:

-   N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-glutamylamide desB30 human     insulin, -   N^(εB29)-ω-carboxy-pentadecanoyl-γ-amino-butanoyl desB30 human     insulin, -   N^(εB29)-ω-carboxy-tetradecanoyl-γ-L-glutamylamide desB30 human     insulin, -   N^(εB29)-ω-carboxy-tridecanoyl-γ-L-glutamylamide desB30 human     insulin, -   N^(εB29)-ω-carboxy-pentadecanoyl-β-alanyl desB30 human insulin, -   N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-aspartylamide desB30 human     insulin, -   N^(εB29)-ω-carboxy-pentadecanoyl-ε-aminohexanoyl desB30 human     insulin, -   N^(εB29)-ω-carboxy-pentadecanoyl-δ-aminopentanoyl desB30 human     insulin, -   N^(εB29)-10-(4-carboxyphenoxy)-decanoyl-γ-L-glutamylamide desB30     human insulin, -   N^(εB29)-4-[11-(4-Carboxyphenyl)undecanoylamino]butyryl desB30 human     insulin, -   N^(εB29)-(3-(3-{4-[3-(7-carboxyheptanoylamino)propoxy]butoxy}propylcarbamoyl)-propionyl-γ-glutamylamide)     desB30 human Insulin, -   N^(εB29)-ω-carboxy-tridecanoyl-γ-amino-butanoyl desB30 human     insulin, -   N^(εB29)-ω-carboxy-undecanoyl-γ-amino-butanoyl desB30 human insulin, -   N^(εB29)-ω-carboxy-tetradecanoyl-γ-amino-butanoyl desB30 human     insulin, -   N^(εB29)-{4-[10-(4-Carboxy-phenoxy)-decanoylamino]-butyryl}desB30     insulin, -   N^(εB29)-{4-[(14-Carboxy-tetradecanoylamino)-methyl]-benzoyl}desB30     insulin, -   N^(εB29)-[16-(4-Carboxy-phenoxy)-hexadecanoyl]desB30 insulin, -   N^(εB29)-{4-[(15-carboxypentadecanoylamino)benzoyl]-desB30 human     insulin and -   N^(εB29)-{4-[(15-Carboxy-pentadecanoylamino)-methyl]-benzoyl}-desB30     insulin

DEFINITIONS

By “insulin analogue” as used herein is meant a polypeptide which has a molecular structure which formally can be derived from the structure of a naturally occurring insulin, for example that of human insulin, by deleting and/or exchanging at least one amino acid residue occurring in the naturally occurring insulin and/or adding at least one amino acid residue. The added and/or exchanged amino acid residues can either be codable amino acid residues or other naturally occurring residues or purely synthetic amino acid residues.

In aspects of the invention a maximum of 17 amino acids have been modified. In aspects of the invention a maximum of 15 amino acids have been modified. In aspects of the invention a maximum of 10 amino acids have been modified. In aspects of the invention a maximum of 8 amino acids have been modified. In aspects of the invention a maximum of 7 amino acids have been modified. In aspects of the invention a maximum of 6 amino acids have been modified. In aspects of the invention a maximum of 5 amino acids have been modified. In aspects of the invention a maximum of 4 amino acids have been modified. In aspects of the invention a maximum of 3 amino acids have been modified. In aspects of the invention a maximum of 2 amino acids have been modified. In aspects of the invention 1 amino acid has been modified.

With “desB30 insulin”, “desB30 human insulin” is meant a natural insulin or an analogue thereof lacking the B30 amino acid residue. Similarly, “desB29desB30 insulin” or “desB29desB30 human insulin” means a natural insulin or an analogue thereof lacking the B29 and B30 amino acid residues.

With “B1”, “A1” etc. is meant the amino acid residue at position 1 in the B-chain of insulin (counted from the N-terminal end) and the amino acid residue at position 1 in the A-chain of insulin (counted from the N-terminal end), respectively. The amino acid residue in a specific position may also be denoted as e.g. PheB1 which means that the amino acid residue at position B1 is a phenylalanine residue.

With “insulin” as used herein is meant human insulin, porcine insulin or bovine insulin with disulfide bridges between CysA7 and CysB7 and between CysA20 and CysB19 and an internal disulfide bridge between CysA6 and CysA11.

By “parent insulin” is meant a naturally occurring insulin such as human insulin or porcine insulin. Alternatively, the parent insulin can be an insulin analogue.

The expression “uncharged” means that no group or groups that would assume a charge at pH interval 4 to 9 are present. For example no free carboxylic acids are present.

With “fatty difunctionalized moiety” is meant a carbon chain of 6 to 32 carbon atoms comprising two functional groups selected from carboxy, amino or hydroxyl.

The term “non-linking amide” is meant to describe an amide function present in a side chain or side group of a residue present in the substituent, such that said amide bond is not used to connect the residues of the substituent together. It should be understood that a residue of the substituent in addition to the non-linking amide can comprise further amide groups, eg. amides that bind to other residues of the substituent.

“Amino acid amide residue” means the alpha-carboxy amide of an amino acid, or if the amino acid contains a carboxylic acid in the side-chain, “amino acid amide” means amide of either the alpha-carboxy group, or amide of the side-chain carboxy group, as specified.

When an insulin derivative according to the invention is stated to be “soluble at physiological pH values” it means that the insulin derivative can be used for preparing insulin compositions that are fully dissolved at physiological pH values. Such favourable solubility may either be due to the inherent properties of the insulin derivative alone or a result of a favourable interaction between the insulin derivative and one or more ingredients contained in the vehicle.

The term “no blunting” as used herein means that when formulated in one formulation both the rapid acting insulin and the acylated insulin has profile of action which is identical or substantially identical with the profile of action, when administering the rapid acting insulin and the acylated insulin in separate formulations.

The expression “high molecular weight insulin” or “hmw” means that the molecular weight of a complex of human insulin, of an insulin analogue or of an insulin derivative is above human serum albumin, above a dodecameric complex of an insulin analogue or of an insulin derivative or more than about 72 kDalton.

The expression “medium molecular weight insulin” or “mmw” means that the molecular weight of a complex of human insulin, of an insulin analogue or of an insulin derivative is from about an insulin hexamer to about an insulin dodecamer between 24 and 80 kDalton.

The expression “low molecular weight insulin” or “lmw” means that the molecular weight of a human insulin, an insulin analogue or an insulin derivative is below 24 kDalton.

The following abbreviations have been used in the specification and examples:

Cv column volume

HPLC High Performance Liquid Chromatography

HSA human serum albumin

LC liquid chromatography

MALDI Matrix Assisted Laser Desorption Ionization

MS mass spectrometry

RT room temperature

SEC size exclusion chromatography

SPA Scitillation Proximity Assay

Tris tris(hydroxymethyl)aminomethane

O.D. optical density=absorbance

X2 monomer AspB9 GluB27 human insulin

DIEA: N,N-diisopropylethylamine

DMF: N,N-dimethylformamide

Sar: Sarcosine (N-methyl-glycine)

tBu: tert-butyl

TSTU: O—(N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate

THF: Tetrahydrofuran

EtOAc: Ethyl acetate

DIPEA: Diisopropylethylamine TEA: triethyl amine

TFA: trifluoracetic acid

DCM: dichloromethane

RT: room temperature

PEG: polyethyleneglycol

GIR: Glucose infusion rate

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law).

All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents.

This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law.

DESCRIPTION OF THE INVENTION

The present invention is based on the recognition that having a substituent in an insulin derivative molecule where only the terminal group is charged, plays an important role for the in vivo duration of action of prolonged-acting insulins, and for the mixability of prolonged-acting insulin with fast-acting insulin with no blunting.

Advantageously, insulin derivatives according to the invention are soluble at physiological pH values, have a potency which is comparable to that of human insulin, and are mixable with fast-acting insulins with no blunting. The individual profiles of action of mixed basal and bolus insulins are retained in formulations containing Zn(II) concentrations of up to or less than approximately 3 Zn(II) per insulin hexamer which limits the risk of precipitations in the formulation, compared to formulations containing more than 3 Zn (II) per insulin hexamer.

The invention is summarized in the following paragraphs:

An insulin derivative having a formula

wherein Ins is a parent insulin moiety and Q₁-Q₂-[CH₂]_(n)—X₁—[CH₂]_(n)-Q₃-[CH₂]_(n)—X₂—[CH₂]_(n)-Q₄-[CH₂]_(n)—X₃—[CH₂]_(n)-Q₅-[CH₂]_(n)—Z is a substituent and where the Ins is attached to the substituent via an amide bond between the α-amino group of the N-terminal amino acid residue of the B chain of Ins or an ε-amino group of a Lys residue present in the A or B chain of Ins and a CO group in Q₁ or Q₂ of the substituent; each n is independently 0, 1, 2, 3, 4, 5 or 6; Q₁ is:

-   -   an amino acid amide of an amino acid with a carboxylic acid in         the side chain, or an amino acid with an uncharged side chain,         which residue forms, with its carboxylic acid group, an amide         group together with the α-amino group of the N-terminal amino         acid residue of the B chain of Ins or together with the ε-amino         group of a Lys residue present in the A or B chain of Ins, or

a chain composed of two, three or four α-amino acid amide or amino acid residues as specified above linked together via amide bonds, which chain—via an amide bond—is linked to the α-amino group of the N-terminal amino acid residue of the B chain of Ins or to the ε-amino group of a Lys residue present in the A or B chain of Ins, or

a bond

Q₂ is:

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—

—CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—;

—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃; or

a bond

-   -   provided that         -   at least one of Q₁ or Q₂ is not a bond, and         -   that Q₂ is not —CO—(CH₂)₂—CO—NH— when n is 0 or 1, X₁ is a             bond and Q₃ is (CH₂CH₂O)₂—, (CH₂CH₂O)₃— or             (CH₂CH₂OCH₂CH₂CH₂CH₂O)— and         -   that if an amine in Q₁ or Q₂ forms a bond with the rest of             the substituent, the amine must be bound to the rest of the             substituent via a carbonyl group;             Q₃, Q₄, and Q₅ independently of each other can be

—(CH₂)_(m)— where m is an integer in the range of 6 to 32;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH₂— groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 32;

—CO—((CR⁵R⁶)₁₋₆—NH—CO)—;

—(CO—(CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃;

—CO—(CH₂)₀₋₃—Ar—(CH₂)₀₋₃— where Ar can be arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R² where R¹ and R², independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃;

(CH₂CH₂O)_(y)—; (CH₂CH₂CH₂O)_(y)—; (CH₂CH₂CH₂CH₂O)_(y)—; (CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)— or (CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)—; —(CH₂OCH₂)_(y)— where y is 1-20;

arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R² where R¹ and R², independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

a chain of the formula —(CH₂)_(s)—Y₁—(Ar)_(v1)—Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)— wherein Ar is defined as above, Y₁-Y₆ independently of each other can be O, S, S═O, SO₂ or a bond; where s, w, t and z independently of each other are zero or an integer from 1 to 10 so that the sum of s, w, t and z is in the range from 4 to 30, and v₁, v₂, and v₃ independently of each other can be zero or 1 with the proviso that Y₁-Y₆ do not link to each other and that the structure —O—(CH₂)₁—O— does not occur; or

a bond;

with the proviso that at least one of Q₃-Q₅ is not a bond;

X₁, X₂ and X₃ are independently of each other

O;

—C═O

-   -   a bond;     -   NCOR¹, where R¹ can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;

with the proviso that

-   -   —X₁, X₂ and X₃ cannot bind to Z and     -   when X₁, X₂ and X₃ are O then X₁, X₂ and X₃ do not bind directly         to O in Q₃, Q₄, and Q₅         and         Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)₂;

—N(CH₂COOH)₂;

—SO₃H

—OSO₃H

—OPO3H₂

—PO₃H₂ or

-tetrazol-5-yl or

—O—W₁,

where W₁ is arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of tetrazo-5-lyl, —COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONR³R⁴ or —SO₂NR³R⁴ where R³ and R⁴ independently of each other can be H, —(CH₂)₁₋₆—SO₃H, or —(CH₂)₁₋₆—O—PO₃H₂; provided that when Z is —O—W₁ then Q₁ must be present; and any Zn²⁺ complex thereof.

2. An insulin derivative having a formula

wherein Ins is a parent insulin moiety and Q₁-Q₂-[CH₂]_(n)—X₁—[CH₂]_(n)-Q₃-[CH₂]_(n)—X₂—[CH₂]_(n)-Q₄-[CH₂]_(n)—X₃—[CH₂]_(n)-Q₅-[CH₂]_(n)—Z is a substituent and where the Ins is attached to the substituent via an amide bond between the α-amino group of the N-terminal amino acid residue of the B chain of Ins or an ε-amino group of a Lys residue present in the A or B chain of Ins and a CO group in Q₁ or Q₂ of the substituent; each n is independently 0, 1, 2, 3, 4, 5 or 6; Q₁ is:

-   -   an amino acid amide of an amino acid with a carboxylic acid in         the side chain, or an amino acid with an uncharged side chain,         which residue forms, with its carboxylic acid group, an amide         group together with the α-amino group of the N-terminal amino         acid residue of the B chain of Ins or together with the ε-amino         group of a Lys residue present in the A or B chain of Ins, or

a chain composed of two, three or four α-amino acid amide or amino acid residues as specified above linked together via amide bonds, which chain—via an amide bond—is linked to the α-amino group of the N-terminal amino acid residue of the B chain of Ins or to the ε-amino group of a Lys residue present in the A or B chain of Ins, or

a bond

Q₂ is:

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—

—CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—;

—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃; or

a bond

-   -   provided that         -   at least one of Q₁ or Q₂ is not a bond, and         -   that Q₂ is not —CO—(CH₂)₂—CO—NH— when n is 0 or 1, X₁ is a             bond and Q₃ is (CH₂CH₂O)₂—, (CH₂CH₂O)₃— or             (CH₂CH₂OCH₂CH₂CH₂CH₂O)— and         -   that if an amine in Q₁ or Q₂ forms a bond with the rest of             the substituent, the amine must be bound to the rest of the             substituent via a carbonyl group;             Q₃, Q₄, and Q₅ independently of each other can be

—(CH₂)_(m)— where m is an integer in the range of 6 to 32;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH₂— groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 32;

—CO—((CR⁵R⁶)₁₋₆—NH—CO)—;

—(CO—(CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃;

—CO—(CH₂)₀₋₃—Ar—(CH₂)₀₋₃— where Ar can be arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R² where R¹ and R² independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃;

(CH₂CH₂O)_(y)—; (CH₂CH₂CH₂O)_(y)—; (CH₂CH₂CH₂CH₂O)_(y)—; (CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)— or (CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)—; —(CH₂OCH₂)_(y)— where y is 1-20;

arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R² where R¹ and R², independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

a chain of the formula

—(CH₂)_(s)—Y₁—(Ar)_(v1)—Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)—

wherein Ar is defined as above, Y₁-Y₆ independently of each other can be O, S, S═O, SO₂ or a bond; where s, w, t and z independently of each other are zero or an integer from 1 to 10 so that the sum of s, w, t and z is in the range from 4 to 30, and v₁, v₂, and v₃ independently of each other can be zero or 1 with the proviso that Y₁-Y₆ do not link to each other and that the structure —O—(CH₂)₁—O— does not occur; or

a bond;

with the proviso that

-   -   at least one of Q₃-Q₅ is not a bond and     -   Q₂ is not —CO—((CH₂)₁₋₆—NH—CO)₁₋₄— when one of Q₃-Q₅ is arylene         or heteroarylene or a chain of the formula         —(CH₂)_(s)—Y₁—(C₆H₄)_(v1)—Y₂—(CH₂)_(w)—Y₃—(C₆H₄)_(v2)—Y₄—(CH₂)_(t)—Y₅—(C₆H₄)_(v3)—Y₆—(CH₂)_(z)—;         X₁, X₂ and X₃ are independently of each other

O;

—C═O

a bond;

NCOR¹, where R¹ can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;

with the proviso that

-   -   —X₁, X₂ and X₃ cannot bind to Z and     -   when X₁, X₂ and X₃ are O then X₁, X₂ and X₃ do not bind directly         to O in Q₃, Q₄, and Q₅         and         Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)₂;

—N(CH₂COOH)₂;

—SO₃H

—OSO₃H

—OPO3H₂

—PO₃H₂ or

-tetrazol-5-yl or

—O—W₁,

where W₁ is arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of tetrazo-5-lyl, —COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONR³R⁴ or —SO₂NR³R⁴ where R³ and R⁴, independently of each other can be H, —(CH₂)₁₋₆—SO₃H, or —(CH₂)₁₋₆—O—PO₃H₂; provided that when Z is —O—W₁ then Q₁ must be present; and any Zn²⁺ complex thereof.

3. An insulin derivative having a formula

wherein Ins is a parent insulin moiety and Q₁-Q₂-[CH₂]_(n)—X₁—[CH₂]_(n)-Q₃-[CH₂]_(n)—X₂—[CH₂]_(n)-Q₄-[CH₂]_(n)—X₃—[CH₂]_(n)-Q₅-[CH₂]_(n)—Z is a substituent and where the Ins is attached to the substituent via an amide bond between the α-amino group of the N-terminal amino acid residue of the B chain of Ins or an ε-amino group of a Lys residue present in the A or B chain of Ins and a CO group in Q₁ or Q₂ of the substituent; each n is independently 0, 1, 2, 3, 4, 5 or 6; Q₁ is:

-   -   an amino acid amide of an amino acid with a carboxylic acid in         the side chain, or an amino acid with an uncharged side chain,         which residue forms, with its carboxylic acid group, an amide         group together with the α-amino group of the N-terminal amino         acid residue of the B chain of Ins or together with the ε-amino         group of a Lys residue present in the A or B chain of Ins, or

a chain composed of two, three or four α-amino acid amide or amino acid residues as specified above linked together via amide bonds, which chain—via an amide bond—is linked to the α-amino group of the N-terminal amino acid residue of the B chain of Ins or to the ε-amino group of a Lys residue present in the A or B chain of Ins, or

a bond

Q₂ is:

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—

—CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—;

—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃; or

a bond

-   -   provided that         -   at least one of Q₁ or Q₂ is not a bond, and         -   that Q₂ is not —CO—(CH₂)₂—CO—NH— when n is 0 or 1, X₁ is a             bond and Q₃ is (CH₂CH₂O)₂—, (CH₂CH₂O)₃— or             (CH₂CH₂OCH₂CH₂CH₂CH₂O)— and         -   that if an amine in Q₁ or Q₂ forms a bond with the rest of             the substituent, the amine must be bound to the rest of the             substituent via a carbonyl group;             Q₃, Q₄, and Q₅ independently of each other can be

—(CH₂)_(m)— where m is an integer in the range of 6 to 32;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH₂— groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 32;

—CO—((CR⁵R⁶)₁₋₆—NH—CO)—;

—(CO—(CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃;

—CO—(CH₂)₀₋₃—Ar—(CH₂)₀₋₃— where Ar can be arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R² where R¹ and R², independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃;

(CH₂CH₂O)_(y)—; (CH₂CH₂CH₂O)_(y)—; (CH₂CH₂CH₂CH₂O)_(y)—; (CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)— or (CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)—; —(CH₂OCH₂)_(y)— where y is 1-20;

arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R², where R¹ and R², independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

a chain of the formula —(CH₂)_(s)—Y₁—(Ar)_(v1)—Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)— wherein Ar is defined as above, Y₁-Y₆ independently of each other can be O, S, S═O, SO₂ or a bond; where s, w, t and z independently of each other are zero or an integer from 1 to 10 so that the sum of s, w, t and z is in the range from 4 to 30, and v₁, v₂, and v₃ independently of each other can be zero or 1 with the proviso that Y₁-Y₆ do not link to each other and that the structure —O—(CH₂)₁—O— does not occur; or

a bond;

with the proviso that

-   -   at least one of Q₃-Q₅ is not a bond and     -   that Q₁ or Q₂ comprises a non-linking amide when one of Q₃-Q₅ is         arylene, heteroarylene or a chain of the formula         —(CH₂)_(s)—Y₁—(C₆H₄)_(v1)—Y₂—(CH₂)_(w)—Y₃—(C₆H₄)_(v2)—Y₄—(CH₂)_(t)—Y₅—(C₆H₄)_(v3)—Y₆—(CH₂)_(z)—.         X₁, X₂ and X₃ are independently of each other

O;

—C═O

a bond;

NCOR¹, where R¹ can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;

with the proviso that

-   -   X₁, X₂ and X₃ cannot bind to Z and     -   when X₁, X₂ and X₃ are O then X₁, X₂ and X₃ do not bind directly         to O in Q₃, Q₄, and Q₅         and         Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)₂;

—N(CH₂COOH)₂;

—SO₃H

—OSO₃H

—OPO3H₂

—PO₃H₂ or

-tetrazol-5-yl or

—O—W₁,

where W₁ is arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of tetrazo-5-lyl, —COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONR³R⁴ or —SO₂NR³R⁴ where R³ and R⁴ independently of each other can be H, —(CH₂)₁₋₆—SO₃H, or —(CH₂)₁₋₆—O—PO₃H₂; provided that when Z is —O—W₁ then Q₁ must be present and any Zn²⁺ complex thereof.

4. An insulin derivative having a formula

wherein Ins is a parent insulin moiety and Q₁-Q₂-[CH₂]_(n)—X₁—[CH₂]_(n)-Q₃-[CH₂]_(n)—X₂—[CH₂]_(n)-Q₄-[CH₂]_(n)—X₃—[CH₂]_(n)-Q₅-[CH₂]_(n)—Z is a substituent and where the Ins is attached to the substituent via an amide bond between the α-amino group of the N-terminal amino acid residue of the B chain of Ins or an ε-amino group of a Lys residue present in the A or B chain of Ins and a CO group in Q₁ or Q₂ of the substituent; each n is independently 0, 1, 2, 3, 4, 5 or 6; Q₁ is:

-   -   an amino acid amide of an amino acid with a carboxylic acid in         the side chain, or an amino acid with an uncharged side chain,         which residue forms, with its carboxylic acid group, an amide         group together with the α-amino group of the N-terminal amino         acid residue of the B chain of Ins or together with the ε-amino         group of a Lys residue present in the A or B chain of Ins, or

a chain composed of two, three or four α-amino acid amide or amino acid residues as specified above linked together via amide bonds, which chain—via an amide bond—is linked to the α-amino group of the N-terminal amino acid residue of the B chain of Ins or to the ε-amino group of a Lys residue present in the A or B chain of Ins, or

a bond

Q₂ is:

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—

—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃; or

a bond

-   -   provided that         -   at least one of Q₁ or Q₂ is not a bond, and         -   that Q₂ is not —CO—(CH₂)₂—CO—NH— when n is 0 or 1, X₁ is a             bond and Q₃ is (CH₂CH₂O)₂—, (CH₂CH₂O)₃— or             (CH₂CH₂OCH₂CH₂CH₂CH₂O)— and         -   that if an amine in Q₁ or Q₂ forms a bond with the rest of             the substituent, the amine must be bound to the rest of the             substituent via a carbonyl group;             -   Q₃, Q₄, and Q₅ independently of each other can be

—(CH₂)_(m)— where m is an integer in the range of 6 to 32;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH₂— groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 32;

—CO—((CR⁵R⁶)₁₋₆—NH—CO)—;

—(CO—(CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃;

—CO—(CH₂)₀₋₃—Ar—(CH₂)₀₋₃— where Ar can be arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R² where R¹ and R², independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃;

(CH₂CH₂O)_(y)—; (CH₂CH₂CH₂O)_(y)—; (CH₂CH₂CH₂CH₂O)_(y)—; (CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)— or (CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)—; —(CH₂OCH₂)_(y)— where y is 1-20;

-   -   arylene or heteroarylene, which may be substituted with one or         two groups selected from the group consisting of —CH₃,         —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R² where R¹ and R²,         independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

a chain of the formula —(CH₂)_(s)—Y₁—(Ar)_(v1)-Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)— wherein Ar is defined as above, Y₁-Y₆ independently of each other can be O, S, S═O, SO₂ or a bond; where s, w, t and z independently of each other are zero or an integer from 1 to 10 so that the sum of s, w, t and z is in the range from 4 to 30, and v₁, v₂, and v₃ independently of each other can be zero or 1 with the proviso that Y₁-Y₆ do not link to each other and that the structure —O—(CH₂)₁—O— does not occur; or

a bond;

with the proviso that at least one of Q₃-Q₅ is not a bond;

X₁, X₂ and X₃ are independently of each other

O;

—C═O

a bond;

NCOR¹, where R¹ can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;

with the proviso that

-   -   —X₁, X₂ and X₃ cannot bind to Z and     -   when X₁, X₂ and X₃ are O then X₁, X₂ and X₃ do not bind directly         to O in Q₃, Q₄, and Q₅         and         Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)₂;

—N(CH₂COOH)₂;

—SO₃H

OSO₃H

—OPO3H₂

—PO₃H₂ or

-tetrazol-5-yl or

—O—W₁,

where W₁ is arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of tetrazo-5-lyl, —COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONR³R⁴ or —SO₂NR³R⁴ where R³ and R⁴, independently of each other can be H, —(CH₂)₁₋₆—SO₃H, or —(CH₂)₁₋₆—O—PO₃H₂; provided that when Z is —O—W₁ then Q₁ must be present and any Zn²⁺ complex thereof.

5. Insulin derivative according to paragraph 1-4, wherein Q₁ is an amino acid amide of an amino acid with a carboxylic acid in the side chain, or an amino acid with an uncharged side chain, the amino acid amide residue or amino acid residue having from 2 to 10 carbon atoms.

6. Insulin derivative according to any of paragraphs 1-5, wherein Q₁ is selected from the group consisting of β-D-Asp-amide, β-L-Asp-amide, γ-L-Glu-amide and γ-D-Glu-amide.

7. Insulin derivative according to paragraphs 1-4, wherein Q₁ is a chain composed of two, three or four α-amino acid amide residues or amino acid residues with an uncharged side chain.

8. Insulin derivative according to paragraphs 1-4 or 7, wherein Q₁ is a chain of two amino acid amide residues selected from the group consisting of β-L-Asp-amide-β-L-Asp-amide, β-L-Asp-amide-γ-L-Glu-amide, γ-L-Glu-amide-γ-L-Glu-amide, γ-L-Glu-amide-β-L-Asp-amide, β-L-Asp-amide-β-D-Asp-amide, β-L-Asp-amide-γ-D-Glu-amide, γ-L-Glu-amide-γ-D-Glu-amide, γ-L-Glu-amide-β-D-Asp-amide, β-D-Asp-amide-β-L-Asp-amide, β-D-Asp-amide-γ-L-Glu-amide, γ-D-Glu-amide-γ-L-Glu-amide, γ-D-Glu-amide-β-L-Asp-amide, β-D-Asp-amide-β-D-Asp-amide, β-D-Asp-amide-γ-D-Glu-amide, γ-D-Glu-amide-γ-D-Glu-amide, γ-D-Glu-amide-β-D-Asp-amide.

9. Insulin derivative according to any of paragraphs 1-8, wherein Q₂ is a bond

10. Insulin derivative according to any of paragraphs 1-3 or 5-8, wherein Q₂ is —CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—.

11. Insulin derivative according to paragraph 10, where Q₂ is selected from the group consisting of —CO—((CH₂)₂—NH—CO)₁—, —CO—((CH₂)₃—NH—CO)₁— and —CO—((CH₂)₄—NH—CO)₁—, —CO—((CH₂)₅—NH—CO)₁—.

12. Insulin derivative according to paragraphs 1, 3 or 10-11, wherein Q₁ is a bond.

13. Insulin derivative according to paragraphs 1-3 wherein

Q₁ is:

an amino acid amide of an amino acid with a carboxylic acid in the side chain, or an amino acid with an uncharged side chain, which residue forms, with its carboxylic acid group, an amide group together with the α-amino group of the N-terminal amino acid residue of the B chain of Ins or together with the ε-amino group of a Lys residue present in the A or B chain of Ins, or

a chain composed of two, three or four α-amino acid amide or amino acid residues as specified above linked together via amide bonds, in which the chain—via an amide bond—is linked to the α-amino group of the N-terminal amino acid residue of the B chain of Ins or to the ε-amino group of a Lys residue present in the A or B chain of Ins, or

a bond

Q₂ is:

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—

—CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—;

—(CO—(CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ can be H, —CH₃, —(CH₂)₁₋₆CH₃; or

a bond

provided that at least one of Q₁ or Q₂ is not a bond and that if an amine in Q₁ or Q₂ forms a bond with the rest of the substituent, the amine must be bound to the rest of the substituent via a carbonyl group;

Q₃ is

—(CH₂)_(m)— where m is an integer in the range of 6 to 32;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH₂— groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 32; or

a divalent hydrocarbon chain of the formula —(CH₂)_(s)C₆H₄(CH₂)_(w)— wherein v and w are integers or one of them is zero so that the sum of s and w is in the range of 6 to 30;

X₁, can be —C═O or a bond;

Q₄, Q₅, X₂ and X₃ are bonds;

All values of n are zero; and

Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)₂;

—N(CH₂COOH)₂;

—SO₃H

—OSO₃H

—OPO3H₂

—PO₃H₂ or

-tetrazol-5-yl

and any Zn²⁺ complex thereof

14. Insulin derivative according to paragraph 13, wherein Q₁ is an amino acid amide of an amino acid with a carboxylic acid in the side chain, or an amino acid with an uncharged side chain, the amino acid amide residue or amino acid residue having from 2 to 10 carbon atoms.

15. Insulin derivative according to any of paragraphs 13-14, wherein Q₁ is selected from the group consisting of β-D-Asp-amide, β-L-Asp-amide, γ-L-Glu-amide and γ-D-Glu-amide.

16. Insulin derivative according to paragraph 13, wherein Q₁ is a chain composed of two, three or four α-amino acid amide residues and/or amino acid residues with an uncharged side chain.

17. Insulin derivative according to paragraphs 13 or 16, wherein Q₁ is a chain of two amino acid amide residues selected from the group consisting of β-L-Asp-amide-β-L-Asp-amide, β-L-Asp-amide-γ-L-Glu-amide, γ-L-Glu-amide-γ-L-Glu-amide, γ-L-Glu-amide-β-L-Asp-amide, β-L-Asp-amide-β-D-Asp-amide, β-L-Asp-amide-γ-D-Glu-amide, γ-L-Glu-amide-γ-D-Glu-amide, γ-L-Glu-amide-β-D-Asp-amide, β-D-Asp-amide-β-L-Asp-amide, β-D-Asp-amide-γ-L-Glu-amide, γ-D-Glu-amide-γ-L-Glu-amide, γ-D-Glu-amide-β-L-Asp-amide, β-D-Asp-amide-β-D-Asp-amide, β-D-Asp-amide-γ-D-Glu-amide, γ-D-Glu-amide-γ-D-Glu-amide, γ-D-Glu-amide-β-D-Asp-amide.

18. Insulin derivative according to any of paragraphs 13-17, wherein Q₂ is a bond.

19. Insulin derivative according to paragraphs 13-17, wherein Q₂ is selected from the group consisting of

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—;

—CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—; or

—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃

20. Insulin derivative according to paragraph 19, wherein Q₂ is selected from the group consisting of —COCH₂OCH₂CONH—, —CO—((CH₂)₂—NH—CO)₁—, —CO—((CH₂)₃—NH—CO)₁—, —CO—((CH₂)₄—NH—CO)₁— and —CO—((CH₂)₅—NH—CO)₁—.

21. Insulin derivative according to paragraphs 13 or 19-21, wherein Q₁ is a bond.

22. Insulin derivative according to any of paragraphs 13-21, wherein X₁ is —C═O.

23. Insulin derivative according to any of paragraphs 13-22, wherein Q₃ is —(CH₂)_(m)— where m is an integer in the range of 6 to 32 or from 8 to 20.

24. Insulin derivative according to paragraph 23, where m is 12, 13, 14, 15 or 16.

25. Insulin derivative according to paragraphs 13-22, wherein Q₃ is a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH₂— groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 32.

26. Insulin derivative according to any of paragraphs 13-25, wherein Z is —COOH.

27. Insulin derivative according to any of paragraphs 13-25, wherein Z is —CH(COOH)₂.

28. An insulin derivative according to any of paragraphs 13-25, wherein Z is —N(CH₂COOH)₂.

29. An insulin derivative according to any of paragraphs 13-25, wherein Z is —SO₃H.

30. An insulin derivative according to any of paragraphs 13-25, wherein Z is —PO₃H.

31. Insulin derivative according to any of paragraphs 13-30 wherein the insulin derivative is selected from the group consisting of

-   N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-glutamylamide desB30 human     insulin, -   N^(εB29)-ω-carboxy-pentadecanoyl-γ-amino-butanoyl desB30 human     insulin, -   N^(εB29)-ω-carboxy-tetradecanoyl-γ-L-glutamylamide desB30 human     insulin, -   N^(εB29)-ω-carboxy-tridecanoyl-γ-L-glutamylamide desB30 human     insulin, -   N^(εB29)-ω-carboxy-pentadecanoyl-β-alanyl desB30 human insulin, -   N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-aspartylamide desB30 human     insulin, -   N^(εB29)-ω-carboxy-pentadecanoyl-ε-aminohexanoyl desB30 human     insulin, -   N^(εB29)-ω-carboxy-pentadecanoyl-δ-aminopentanoyl desB30 human     insulin, -   N^(εB29)-ω-carboxy-tridecanoyl-γ-amino-butanoyl desB30 human insulin -   N^(εB29)-ω-carboxy-undecanoyl-δ-amino-butanoyl desB30 human insulin     and -   N^(εB29)-ω-carboxy-tetradecanoyl-γ-amino-butanoyl desB30 human     insulin

32. Insulin derivative according to paragraph 2 or 4, wherein

Q₃ is:

—CO—((CR⁵R⁶)₁₋₆—NH—CO)—;

—(CO—(CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃;

—CO—(CH₂)₀₋₃—Ar—(CH₂)₀₋₃— where Ar can be arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R² where R¹ and R² independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

a bond

Q₄ is:

—(CH₂)_(m)— where m is an integer from 4 to 22;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH₂— groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 22;

arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R², where R¹ and R², independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

a chain of the formula —(CH₂)_(s)—Y₁—(Ar)_(v1)—Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)— wherein Ar is defined as above, Y₁-Y₆ independently of each other can be O, S, S═O, SO₂ or a bond; where s, w, t and z independently of each other are zero or an integer from 1 to 10 so that the sum of s, w, t and z is in the range from 4 to 30, and v₁, v₂, and v₃ independently of each other can be zero or 1 with the proviso that Y₁-Y₆ do not link to each other and that the structure —O—(CH₂)₁—O— does not occur; X₁ is:

O;

—C═O

NCOR¹, where R¹ can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

-   -   where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;     -   with the proviso that when X₁ is O, then X₁ does not bind         directly to O in Q₄;         X₂, X₃ and Q₅ are bonds;         All values of n are zero; and         Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)₂;

—N(CH₂COOH)₂;

—SO₃H

—OSO₃H

—OPO3H₂

—PO₃H₂ or

-tetrazol-5-yl or

—O—W₁,

where W₁ is arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of tetrazo-5-lyl, —COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONR³R⁴ or —SO₂NR³R⁴, where R³ and R⁴ independently of each other can be H, —(CH₂)₁₋₆—SO₃H, or —(CH₂)₁₋₆—O—PO₃H₂; and any Zn²⁺ complex thereof.

33. Insulin derivative according to paragraphs 2, 4 or 32, wherein Q₁ is an amino acid amide of an amino acid with a carboxylic acid in the side chain, or an amino acid with an uncharged side chain, the amino acid amide residue or amino acid residue having from 2 to 10 carbon atoms.

34. Insulin derivative according to paragraphs 2, 4 or 33, wherein Q₁ is selected from the group consisting of β-D-Asp-amide, β-L-Asp-amide, γ-L-Glu-amide and γ-D-Glu-amide.

35. Insulin derivative according to paragraph 2, 4 or 34, wherein Q₁ is a chain composed of two, three or four α-amino acid amide residues or amino acid residues with an uncharged side chain.

36. Insulin derivative according to paragraphs 2, 4 or 35, wherein Q₁ is a chain of two amino acid amide residues selected from the group consisting of β-L-Asp-amide-β-L-Asp-amide, β-L-Asp-amide-γ-L-Glu-amide, γ-L-Glu-amide-γ-L-Glu-amide, γ-L-Glu-amide-β-L-Asp-amide, β-L-Asp-amide-β-D-Asp-amide, β-L-Asp-amide-γ-D-Glu-amide, γ-L-Glu-amide-γ-D-Glu-amide, γ-L-Glu-amide-β-D-Asp-amide, β-D-Asp-amide-β-L-Asp-amide, β-D-Asp-amide-γ-L-Glu-amide, γ-D-Glu-amide-γ-L-Glu-amide, γ-D-Glu-amide-β-L-Asp-amide, β-D-Asp-amide-β-D-Asp-amide, β-D-Asp-amide-γ-D-Glu-amide, γ-D-Glu-amide-γ-D-Glu-amide, γ-D-Glu-amide-β-D-Asp-amide.

37. Insulin derivative according to any of paragraphs 2, 4 or 32-36, wherein Q₂ is a bond.

38. Insulin derivative according to paragraphs 2, 4 or 32-36, where Q₂ is —CO—CH₂—(CHCONH₂)—NH—CO; —CO—(CH₂)₂—(CHCONH₂)—NH—CO)— or —CO—(CH₂)₃—(CHCONH₂)—NH—CO)—.

39. Insulin derivative according to paragraph 2, 4 or 38, wherein Q₁ is a bond.

40. Insulin derivative according to paragraphs 32-39, wherein Q₃ is —(CH₂)_(s)—Y₁—(Ar)_(v1)—Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)—

wherein Ar is defined as above, Y₁-Y₆ independently of each other can be O, S, S═O, SO₂ or a bond; where s, w, t and z independently of each other are zero or an integer from 1 to 10 so that the sum of s, w, t and z is in the range from 4 to 30, and v₁, v₂, and v₃ independently of each other can be zero or 1 with the proviso that Y₁-Y₆ do not link to each other and that the structure —O—(CH₂)₁—O— does not occur.

41. Insulin derivative according to any of paragraph 40, wherein at least two of v₁, v₂, or v₃ are zero.

42. Insulin derivative according to any of paragraphs 40-41, wherein Y₁-Y₆ are bonds.

43. Insulin derivative according to any of paragraphs 40-41, wherein at least one of Y₁-Y₆ are O or S.

44. Insulin derivative according to any of paragraphs 40-41 or 43, wherein Y₁ is O or S and v₁ is one.

45. Insulin derivative according to any of paragraphs 40-44, wherein s is 6, 7, 8, 9 or 10.

46. Insulin derivative according to any of paragraphs 40-45, wherein X₁, X₂, X₃, Q₄, and Q₅ are bonds and Ar is C₆H₄.

47. Insulin derivative according to any of paragraphs 32-46, wherein n is zero.

48. Insulin derivative according to any of paragraphs 32-47, wherein Z is —COOH.

49. Insulin derivative according to any of paragraphs 32-47, wherein Z is —CH(COOH)₂.

50. An insulin derivative according to any of paragraphs 32-47, wherein Z is —N(CH₂COOH)₂.

51. An insulin derivative according to any of paragraphs 32-47, wherein Z is —SO₃H.

52. An insulin derivative according to any of paragraphs 32-47, wherein Z is —PO₃H.

53. An insulin derivative according to any of paragraphs 32-47, wherein Z is —O—W₁, where W₁ is arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of tetrazo-5-lyl, —COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONR¹³R¹⁴ or —SO₂NR¹³R¹⁴ where R¹³ and R¹⁴, independently of each other can be H, —(CH₂)₁₋₆—SO₃H, or —(CH₂)₁₋₆—O—PO₃H₂.

54. Insulin derivative according to any of paragraphs 32-53 wherein the insulin derivative is N^(εB29)-10-(4-carboxyphenoxy)-decanoyl-γ-L-glutamylamide desB30 human insulin.

55. Insulin derivative according to paragraph 1, wherein Q₁ is an amino acid amide of an amino acid with a carboxylic acid in the side chain, or an amino acid with an uncharged side chain, the amino acid amide residue or amino acid residue having from 2 to 10 carbon atoms.

56. Insulin derivative according to any of paragraphs 1 or 55, wherein Q₁ is selected from the group consisting of β-D-Asp-amide, β-L-Asp-amide, γ-L-Glu-amide and γ-D-Glu-amide.

57. Insulin derivative according to paragraph 1, wherein Q₁ is a chain composed of two, three or four α-amino acid amide residues or amino acid residues with an uncharged side chain.

58. Insulin derivative according to paragraphs 1 or 57, wherein Q₁ is a chain of two amino acid amide residues selected from the group consisting of β-L-Asp-amide-β-L-Asp-amide, β-L-Asp-amide-γ-L-Glu-amide, γ-L-Glu-amide-γ-L-Glu-amide, γ-L-Glu-amide-β-L-Asp-amide, β-L-Asp-amide-β-D-Asp-amide, β-L-Asp-amide-γ-D-Glu-amide, γ-L-Glu-amide-γ-D-Glu-amide, γ-L-Glu-amide-β-D-Asp-amide, β-D-Asp-amide-β-L-Asp-amide, β-D-Asp-amide-γ-L-Glu-amide, γ-D-Glu-amide-γ-L-Glu-amide, γ-D-Glu-amide-β-L-Asp-amide, β-D-Asp-amide-β-D-Asp-amide, β-D-Asp-amide-γ-D-Glu-amide, γ-D-Glu-amide-γ-D-Glu-amide, γ-D-Glu-amide-β-D-Asp-amide.

59. Insulin derivative according to any of paragraphs 1 or 55-58, wherein Q₂ is a bond.

60. Insulin derivative according to any of paragraphs 1 or 55-58, wherein Q₂ is selected from the group consisting of —CO—(CR⁵R⁶)₁₋₆—NH—CO— and —(CO—(CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃;

61. Insulin derivative according to paragraphs 1 or 60, where Q₂ is —CO—((CH₂)₃—NH—CO)—, —CO—CH₂—(CHCONH₂)—NH—CO; —CO—(CH₂)₂—(CHCONH₂)—NH—CO)— or —CO—(CH₂)₃—(CHCONH₂)—NH—CO)—

62. Insulin derivative according to paragraphs 1 or 60-61, wherein Q₁ is a bond.

63. Insulin derivative according to paragraph 55-62, wherein Q₃ is —(CH₂)_(s)—Y₁—(Ar)_(v1)—Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)—

wherein Ar is defined as above, Y₁-Y₆ independently of each other can be O, S, S═O, SO₂ or a bond; where s, w, t and z independently of each other are zero or an integer from 1 to 10 so that the sum of s, w, t and z is in the range from 4 to 30, and v₁, v₂, and v₃ independently of each other can be zero or 1 with the proviso that Y₁-Y₆ do not link to each other and that the structure —O—(CH₂)₁—O— does not occur.

64. Insulin derivative according to paragraph 63, wherein at least two of v₁, v₂, or v₃ are zero.

65. Insulin derivative according to any of paragraphs 63-64, wherein Y₁-Y₆ are bonds.

66. Insulin derivative according to any of paragraphs 63-64, wherein at least one of Y₁—Y₆ are O or S.

67. Insulin derivative according to any of paragraphs 63-64 or 66, wherein Y₁ is O or S and v₁ is one.

68. Insulin derivative according to any of paragraphs 63-67, wherein s is 6, 7, 8, 9 or 10.

69. Insulin derivative according to any of paragraphs 55-68, wherein X₁, X₂, X₃, Q₄, and Q₅ are bonds and Ar is C₆H₄.

70. Insulin derivative according to any of paragraphs 55-69, wherein n is zero.

71. Insulin derivative according to any of paragraphs 55-70, wherein Z is —COOH.

72. Insulin derivative according to any of paragraphs 55-70, wherein Z is —CH(COOH)₂.

73. An insulin derivative according to any of paragraphs 55-70, wherein Z is —N(CH₂COOH)₂.

74. An insulin derivative according to any of paragraphs 55-70, wherein Z is —SO₃H.

75. An insulin derivative according to any of paragraphs 55-70, wherein Z is —PO₃H.

76. An insulin derivative according to any of paragraphs 55-70, wherein Z is —O—W₁, where W₁ is arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of tetrazo-5-lyl, —COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONR¹³R¹⁴ or —SO₂NR¹³R¹⁴ where R¹³ and R¹⁴, independently of each other can be H, —(CH₂)₁₋₆—SO₃H, or —(CH₂)₁₋₆—O—PO₃H₂.

77. Insulin derivative according to any of paragraphs 1 or 55-70 wherein the insulin derivative are selected from the group consisting of N^(εB29)-10-(4-carboxyphenoxy)-decanoyl-γ-L-glutamylamide desB30 human insulin, N^(εB29)-4-[11-(4-Carboxyphenyl)undecanoylamino]butyryl desB30 human insulin.

78. Insulin derivative according to paragraph 3, wherein Q₁ is an amino acid amide of an amino acid with a carboxylic acid in the side chain having from 2 to 10 carbon atoms.

79. Insulin derivative according to any of paragraphs 3 or 77, wherein Q₁ is selected from the group consisting of β-D-Asp-amide, β-L-Asp-amide, γ-L-Glu-amide and γ-D-Glu-amide.

80. Insulin derivative according to paragraphs 3, wherein Q₁ is a chain of two amino acid amide residues selected from the group consisting of β-L-Asp-amide-β-L-Asp-amide, β-L-Asp-amide-γ-L-Glu-amide, γ-L-Glu-amide-γ-L-Glu-amide, γ-L-Glu-amide-β-L-Asp-amide, β-L-Asp-amide-β-D-Asp-amide, β-L-Asp-amide-γ-D-Glu-amide, γ-L-Glu-amide-γ-D-Glu-amide, γ-L-Glu-amide-β-D-Asp-amide, β-D-Asp-amide-β-L-Asp-amide, β-D-Asp-amide-γ-L-Glu-amide, γ-D-Glu-amide-γ-L-Glu-amide, γ-D-Glu-amide-β-L-Asp-amide, β-D-Asp-amide-β-D-Asp-amide, β-D-Asp-amide-γ-D-Glu-amide, γ-D-Glu-amide-γ-D-Glu-amide, γ-D-Glu-amide-β-D-Asp-amide.

81. Insulin derivative according to any of paragraphs 3 or 77-79, wherein Q₂ is a bond.

82. Insulin derivative according to paragraphs 3 or 77-80, wherein Q₂ is selected from the group consisting of

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—;

—CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—; or

—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ provided that Q₂ comprises a non-linking amide if Q₁ is a bond.

83. Insulin derivative according to paragraphs 3 or 82, wherein Q₁ is a bond.

84. Insulin derivative according to paragraph 78-83, wherein Q₃ is —(CH₂)_(s)—Y₁—(Ar)_(v1)—Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)—

wherein Ar is defined as above, Y₁-Y₆ independently of each other can be O, S, S═O, SO₂ or a bond; where s, w, t and z independently of each other are zero or an integer from 1 to 10 so that the sum of s, w, t and z is in the range from 4 to 30, and v₁, v₂, and v₃ independently of each other can be zero or 1 with the proviso that Y₁-Y₆ do not link to each other and that the structure —O—(CH₂)₁—O— does not occur.

85. Insulin derivative according to paragraph 84, wherein at least two of v₁, v₂, or v₃ are zero.

86. Insulin derivative according to any of paragraphs 84-85, wherein Y₁-Y₆ are bonds.

87. Insulin derivative according to any of paragraphs 84-85, wherein at least one of Y₁—Y₆ are O or S.

88. Insulin derivative according to any of paragraphs 84-85 or 87, wherein Y₁ is O or S and v₁ is one.

89. Insulin derivative according to any of paragraphs 84-88, wherein s is 6, 7, 8, 9 or 10.

90. Insulin derivative according to any of paragraphs 78-89, wherein X₁, X₂, X₃, Q₄, and Q₅ are bonds and Ar is C₆H₄.

91. Insulin derivative according to any of paragraphs 78-90, wherein n is zero.

92. Insulin derivative according to any of paragraphs 78-91, wherein Z is —COOH.

93. Insulin derivative according to any of paragraphs 78-91, wherein Z is —CH(COOH)₂.

94. An insulin derivative according to any of paragraphs 78-91, wherein Z is —N(CH₂COOH)₂.

95. An insulin derivative according to any of paragraphs 78-91, wherein Z is —SO₃H.

96. An insulin derivative according to any of paragraphs 78-91, wherein Z is —PO₃H.

97. An insulin derivative according to any of paragraphs 78-91, wherein Z is —O—W₁, where W₁ is arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of tetrazo-5-lyl, —COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONR¹³R¹⁴ or —SO₂NR¹³R¹⁴ where R¹³ and R¹⁴ independently of each other can be H, —(CH₂)₁₋₆—SO₃H, or —(CH₂)₁₋₆—O—PO₃H₂.

98. Insulin derivative according to any of paragraphs 3 or 78-97, wherein the insulin derivative are selected from the group consisting of:

-   N^(εB29)-10-(4-carboxyphenoxy)-decanoyl-γ-L-glutamylamide desB30     human insulin, -   N^(εB29)-4-[11-(4-Carboxyphenyl)undecanoylamino]butyryl desB30 human     insulin and -   N^(εB29)-{4-[10-(4-Carboxy-phenoxy)-decanoylamino]-butyryl} desB30     insulin

99. Insulin derivative according to paragraphs 1 or 2, wherein Q₁ is an amino acid amide of an amino acid with a carboxylic acid in the side chain, or an amino acid with an uncharged side chain, the amino acid amide residue or amino acid residue having from 2 to 10 carbon atoms.

100. Insulin derivative according to any of paragraphs 1, 2 or 99, wherein Q₁ is selected from the group consisting of β-D-Asp-amide, β-L-Asp-amide, γ-L-Glu-amide and γ-D-Glu-amide.

101. Insulin derivative according to paragraph 1 or 2, wherein Q₁ is a chain composed of two, three or four α-amino acid amide or amino acid residues with uncharged side chains.

102. Insulin derivative according to paragraphs 1, 2 or 101, wherein Q₁ is a chain of two amino acid amide residues selected from the group consisting of β-L-Asp-amide-β-L-Asp-amide, β-L-Asp-amide-γ-L-Glu-amide, γ-L-Glu-amide-γ-L-Glu-amide, γ-L-Glu-amide-β-L-Asp-amide, β-L-Asp-amide-β-D-Asp-amide, β-L-Asp-amide-γ-D-Glu-amide, γ-L-Glu-amide-γ-D-Glu-amide, γ-L-Glu-amide-β-D-Asp-amide, β-D-Asp-amide-β-L-Asp-amide, β-D-Asp-amide-γ-L-Glu-amide, γ-D-Glu-amide-γ-L-Glu-amide, γ-D-Glu-amide-β-L-Asp-amide, β-D-Asp-amide-β-D-Asp-amide, β-D-Asp-amide-γ-D-Glu-amide, γ-D-Glu-amide-γ-D-Glu-amide, γ-D-Glu-amide-β-D-Asp-amide.

103. Insulin derivative according to any of paragraphs 99-102, wherein Q₂ is a bond

104. Insulin derivative according to any of paragraphs 1, 2 or 99-102, wherein Q₂ is selected from the group consisting of —CO—((CR⁵R⁶)₁₋₆—NH—CO)— and —(CO—(CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃;

105. Insulin derivative according to any of paragraphs 1, 2 or 99-104, wherein Q₂ is —CO—CH₂—(CHCONH₂)—NH—CO; —CO—(CH₂)₂—(CHCONH₂)—NH—CO)— or —CO—(CH₂)₃—(CHCONH₂)—NH—CO)—

106. Insulin derivative according to paragraphs 1, 2 or 104-105, wherein Q₁ is a bond.

107. Insulin derivative according any of paragraphs 99-106, wherein one of Q₃, Q₄, or Q₅ is —(CH₂)_(m)— where m is an integer in the range of 1 to 32 or 1-12.

108. Insulin derivative according to paragraph 107, wherein m is 4, 5, 6, 8, 9, 10 or 11.

109. Insulin derivative according to any of paragraphs 99-108, wherein one of Q₃, Q₄, or Q₅ is (CH₂CH₂O)_(y)—; (CH₂CH₂CH₂O)_(y)—; (CH₂CH₂CH₂CH₂O)_(y)—; (CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)— or (CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)—; —(CH₂OCH₂)_(y)— where y is 1-20.

110. Insulin derivative according to paragraph 109, wherein one of Q₃, Q₄, or Q₅ is (CH₂CH₂O)_(y)— or (CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y) wherein y is in the range of 1-12, 2-4 or 2-3

111. Insulin derivative according to any of paragraphs 109-110, wherein y is 1.

112. Insulin derivative according to any of paragraphs 99-111, wherein X₁, X₂ and

X₃ are independently of each other

O;

—C═O

a bond;

NCOR¹, where R¹ can be H, —CH₃ or —(CH)₁₋₆—CH₃; or

where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;

113. Insulin derivative according to any of paragraphs 99-112, wherein X₁, X₂ and X₃ are:

where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;

114. Insulin derivative according to any of paragraphs 99-113, wherein X₁ is

where R is H and wherein X₂ is

where R is H.

115. Insulin derivative according to any of paragraphs 99-114, wherein n is 0, 1, 2 or 3.

116. Insulin derivative according to any of paragraphs 99-115, wherein Z is —COOH.

117. Insulin derivative according to any of paragraphs 99-115, wherein Z is —CH(COOH)₂.

118. An insulin derivative according to any of paragraphs 99-115, wherein Z is —N(CH₂COOH)₂.

119. An insulin derivative according to any of paragraphs 99-115, wherein Z is —SO₃H.

120. An insulin derivative according to any of paragraphs 99-115, wherein Z is —PO₃H.

121. Insulin derivative according to any of paragraphs 1-2 or 99-120 wherein the insulin derivative is

-   N^(εB29)-(3-(3-{4-[3-(7-carboxyheptanoylamino)propoxy]butoxy}propylcarbamoyl)-propionyl-γ-glutamylamide)     desB30 human Insulin.

In one aspect of the invention Q₁ is an amino acid amide of an amino acid with a carboxylic acid in the side chain, or an amino acid with an uncharged side chain, the amino acid amide residue or amino acid residue having from 2 to 10 carbon atoms.

Q₁ can also be a chain composed of two, three or four amino acid amide of an amino acid with a carboxylic acid in the side chain and/or an amino acid with an uncharged side chain The chain of amino acids may comprise at least one amino acid containing an amide. Thus, Q₁ can, for example, be selected from the group comprising β-L-Asp-amide, β-D-Asp-amide, γ-L-Glu-amide and γ-D-Glu-amide.

In one aspect Q₁ is a chain composed of two uncharged amino acid residues, which has from 4 to 10 carbon atoms. The amino acids can comprise an amide. Examples of such amino acid residues are β-L-Asp-amide-β-L-Asp-amide, β-L-Asp-amide-γ-L-Glu-amide, γ-L-Glu-amide-γ-L-Glu-amide, γ-L-Glu-amide-β-L-Asp-amide, β-L-Asp-amide-β-D-Asp-amide, β-L-Asp-amide-γ-D-Glu-amide, γ-L-Glu-amide-γ-D-Glu-amide, γ-L-Glu-amide-β-D-Asp-amide, β-D-Asp-amide-β-L-Asp-amide, β-D-Asp-amide-γ-L-Glu-amide, γ-D-Glu-amide-γ-L-Glu-amide, γ-D-Glu-amide-β-L-Asp-amide, β-D-Asp-amide-β-D-Asp-amide, β-D-Asp-amide-γ-D-Glu-amide, γ-D-Glu-amide-γ-D-Glu-amide, γ-D-Glu-amide-β-D-Asp-amide,

In one aspect of the invention Q₁ is a chain composed of three amino acid residues, independently having from 4 to 10 carbon atoms, where at least one of the amino acid residues of the chain being selected from the group of residues having an amide. The combination of the three amino acid amides can be any combination of β-L-Asp-amide, β-D-Asp-amide, γ-L-Glu-amide and γ-D-Glu-amide, which means that 64 different combinations are possible.

In a further aspect, Q₁ is a chain composed of four amino acid residues, independently having from 4 to 10 carbon atoms, where at least one of the amino acid residues of the chain being selected from the group of residues having an amide. The combination of the four amino acid amides can be any combination of β-L-Asp-amide, β-D-Asp-amide, γ-L-Glu-amide and γ-D-Glu-amide, which means that 256 different combinations are possible.

122. An insulin derivative having a substituent attached to desB30 human insulin at either the α-amino group of the N-terminal amino acid residue of the B chain or at an ε-amino group of a Lys residue present in the A or B chain of desB30 human insulin, which substituent comprises at least one fatty difunctionalized moiety with about 6 to about 32 carbon atoms and an uncharged linker of the formula —CO—((CH₂)₁₋₆—NH—CO)₁₋₄ which links the fatty difunctionalized moiety to desB30 human insulin, wherein the fatty difunctionalized moiety comprises an aromatic group.

123. Insulin derivative according to paragraph 122, wherein the uncharged linker is selected from the group consisting of: —CO—CH₂—NH—CO, —CO—(CH₂)₂—NH—CO, —CO—(CH₂)₃—NH—CO and —CO—(CH₂)₁—NH—CO.

124. Insulin derivative according to paragraphs 122 or 123, wherein the fatty difunctionalized moiety is

T-(CH₂)_(s)—Y₁—(Ar)_(v1)—Y₂—(CH₂)_(w)—Y₃—(Ar)_(v2)—Y₄—(CH₂)_(t)—Y₅—(Ar)_(v3)—Y₆—(CH₂)_(z)-T wherein

Ar can be arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —CH₃, —(CH)₁₋₆—CH₃, —CONR¹R² or —SO₂NR¹R² where R¹ and R², independently of each other can be H, —CH₃ or —(CH)₁₋₆—CH₃;

Y₁-Y₆ independently of each other can be O, S, S═O, SO₂ or a bond;

s, w, t and z independently of each other are zero or an integer from 1 to 10 so that the sum of s, w, t and z is in the range from 4 to 30,

v₁, v₂, and v₃ independently of each other can be zero or 1 with the proviso that Y₁-Y₆ do not link to each other and that the structure —O—(CH₂)₁—O— does not occur; and

T is a functional group selected from carboxy, amino or hydroxyl groups.

125. Insulin derivative according to paragraphs 122-124, wherein the insulin derivative is N^(εB29)-4-[11-(4-Carboxyphenyl)undecanoylamino]butyryl desB30 human insulin.

126. An insulin derivative having a substituent attached to a parent insulin moiety at either the α-amino group of the N-terminal amino acid residue of the B chain or at an ε-amino group of a Lys residue present in the A or B chain of the parent insulin moiety, which substituent comprises at least one fatty difunctionalized moiety with about 6 to about 32 carbon atoms and an uncharged linker which links the fatty difunctionalized moiety to the parent insulin, provided that the uncharged linker does not comprise —(CO—(CH₂)₁₋₆—NH—CO)₁₋₄— if the insulin substituent comprises an aromatic group and that the substituent does not comprise one or more residues selected from the group of —CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₂—, —CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂O)₂—, —CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₃— and —CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂OCH₂CH₂CH₂CH₂O)—.

127. An insulin derivative having a substituent attached to a parent insulin moiety at either the α-amino group of the N-terminal amino acid residue of the B chain or at an ε-amino group of a Lys residue present in the A or B chain of the parent insulin moiety, which substituent comprises at least one fatty difunctionalized moiety with about 6 to about 32 carbon atoms and an uncharged linker which links the fatty difunctionalized moiety to the parent insulin, provided that the substituent does not comprise one or more residues selected from the group of —CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₂—, —CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂O)₂—, —CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₃— and —CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂OCH₂CH₂CH₂CH₂O)—.

128. An insulin derivative having a substituent attached to a parent insulin moiety at either the α-amino group of the N-terminal amino acid residue of the B chain or at an ε-amino group of a Lys residue present in the A or B chain of the parent insulin moiety, which substituent comprises at least one fatty difunctionalized moiety with about 6 to about 32 carbon atoms and an uncharged linker which links the fatty difunctionalized moiety to the parent insulin, provided that the substituent does not comprise one or more residues selected from the group of —CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₂—, —CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂O)₂—, —CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₃— and —CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂OCH₂CH₂CH₂CH₂O)— characterized by the uncharged linker being selected from the group consisting of

-   -   an amino acid amide of an amino acid with a carboxylic acid in         the side chain, or an amino acid with an uncharged side chain,         which residue forms, with its carboxylic acid group, an amide         group together with the α-amino group of the N-terminal amino         acid residue of the B chain of Ins or together with the ε-amino         group of a Lys residue present in the A or B chain of Ins, or

a chain composed of two, three or four α-amino acid amide or amino acid residues as specified above linked together via amide bonds, which chain—via an amide bond—is linked to the α-amino group of the N-terminal amino acid residue of the B chain of Ins or to the ε-amino group of a Lys residue present in the A or B chain of Ins

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—; or

—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃

provided that if an amine in the uncharged linker forms a bond with the rest of the substituent, the amine must be bound to the rest of the substituent via a carbonyl group.

129. An insulin derivative having a substituent attached to a parent insulin moiety at either the α-amino group of the N-terminal amino acid residue of the B chain or at an ε-amino group of a Lys residue present in the A or B chain of the parent insulin moiety, which substituent comprises at least one fatty difunctionalized moiety with about 6 to about 32 carbon atoms and an uncharged linker which links the fatty difunctionalized moiety to the parent insulin, provided that the uncharged linker does not comprise —(CO—(CH₂)₁₋₆—NH—CO)₁₋₄— if the insulin substituent comprises an aromatic group and that the substituent does not comprise one or more residues selected from the group of —CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₂—, —CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂O)₂—, —CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₃— and —CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂OCH₂CH₂CH₂CH₂O)— characterized by the uncharged linker being selected from the group consisting of

-   -   an amino acid amide of an amino acid with a carboxylic acid in         the side chain, or an amino acid with an uncharged side chain,         which residue forms, with its carboxylic acid group, an amide         group together with the α-amino group of the N-terminal amino         acid residue of the B chain of Ins or together with the ε-amino         group of a Lys residue present in the A or B chain of Ins, or

a chain composed of two, three or four α-amino acid amide or amino acid residues as specified above linked together via amide bonds, which chain—via an amide bond—is linked to the α-amino group of the N-terminal amino acid residue of the B chain of Ins or to the ε-amino group of a Lys residue present in the A or B chain of Ins,

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—

—CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—; or

—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃,

provided that if an amine in the uncharged linker forms a bond with the rest of the substituent, the amine must be bound to the rest of the substituent via a carbonyl group.

130. An insulin derivative having a substituent attached to a parent insulin moiety at either the α-amino group of the N-terminal amino acid residue of the B chain or at an ε-amino group of a Lys residue present in the A or B chain of the parent insulin moiety, which substituent comprises at least one fatty difunctionalized moiety with about 6 to about 32 carbon atoms and an uncharged linker which links the fatty difunctionalized moiety to the parent insulin, provided that the substituent does not comprise one or more residues selected from the group of —CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₂—, —CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂O)₂—, —CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₃— and —CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂OCH₂CH₂CH₂CH₂O)— characterized by the uncharged linker being selected from the group consisting of

-   -   an amino acid amide of an amino acid with a carboxylic acid in         the side chain, or an amino acid with an uncharged side chain,         which residue forms, with its carboxylic acid group, an amide         group together with the α-amino group of the N-terminal amino         acid residue of the B chain of Ins or together with the ε-amino         group of a Lys residue present in the A or B chain of Ins, or

a chain composed of two, three or four α-amino acid amide or amino acid residues as specified above linked together via amide bonds, which chain—via an amide bond—is linked to the α-amino group of the N-terminal amino acid residue of the B chain of Ins or to the ε-amino group of a Lys residue present in the A or B chain of Ins,

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—COCH₂OCH₂CONH—

—CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄—; or

—CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃

provided that if an amine in the uncharged linker forms a bond with the rest of the substituent, the amine must be bound to the rest of the substituent via a carbonyl group.

131. An insulin derivative having a substituent attached to a parent insulin moiety at either the α-amino group of the N-terminal amino acid residue of the B chain or at an ε-amino group of a Lys residue present in the A or B chain of the parent insulin moiety, which substituent comprises at least one fatty difunctionalized moiety with about 6 to about 32 carbon atoms and an uncharged linker which links the fatty difunctionalized moiety to the parent insulin, provided that a non-linking amide is present in the linker if the substituent comprises an aromatic group and that the substituent does not comprise one or more residues selected from the group of —CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₂—, —CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂O)₂—, —CO—(CH₂)₂—CO—NH—(CH₂CH₂O)₃— and —CO—(CH₂)₂—CO—NH—CH₂—(CH₂CH₂OCH₂CH₂CH₂CH₂O)—.

132. An insulin derivative having a substituent attached to a parent insulin moiety at either the α-amino group of the N-terminal amino acid residue of the B chain or at an ε-amino group of a Lys residue present in the A or B chain of the parent insulin moiety, which substituent comprises at least one fatty difunctionalized moiety with about 6 to about 32 carbon atoms and an uncharged linker which links the fatty difunctionalized moiety to the parent insulin, provided that the fatty difunctionalized moiety is an aliphatic chain, an non-linking amide is present in the linker.

133. Insulin derivative according to paragraph 121-129, wherein the substituent is attached to the α-amino group of the N-terminal amino acid residue of the B chain of the parent insulin.

134. Insulin derivative according to paragraph 121-129, wherein the substituent is attached to the ε-amino group of a Lys residue present in the A or B chain of the parent insulin.

135. Insulin derivative according to paragraphs 121-129 or 131 wherein the substituent is attached to the ε-amino group of the Lys residue in position B29 present in the B chain of the parent insulin.

136. Insulin derivative according to paragraph 132, wherein the substituent is attached to the ε-amino group of the Lys residue in position B29 in desB30 human insulin

137. Insulin derivative according to any of paragraphs 121-129, wherein the linker comprises an amide or an N-substituted amide of the formula —CONR⁷R⁸ where R⁷ and R⁸, independently of each other can be hydrogen, methyl, ethyl, propyl or isopropyl.

138. Insulin derivative according to paragraph 134, where R⁷ and R⁸ are hydrogen.

139. Insulin derivative according to paragraph 121-129, wherein the linker comprises a non-linking amide.

140. Insulin derivative according to any of paragraphs 121-129, wherein the substituent comprises one or more residues of ethyleneglycol, propyleneglycol and/or butyleneglycol containing independently at each termini a group selected from —NH₂ and —COOH, which is used to connect the individual components of the substituent.

141. Insulin derivative according to paragraphs 121-129, wherein the substituent comprises at least one aromatic group.

142. Insulin derivative according to any of the paragraphs 1-141, wherein the parent insulin is human insulin or porcine insulin.

143 Insulin derivative according to any of the paragraphs 1-141, wherein the parent insulin is an insulin analogue.

144. Insulin derivative according to any of paragraphs 143, wherein the amino acid residue at position B30 of the parent insulin is Lys or has been deleted.

145. Insulin derivative according to paragraphs 143-44, wherein the parent insulin is desB30 human insulin.

146. Insulin derivative according to any of paragraphs 143-145, wherein the amino acid residue at position B1 of the parent insulin has been deleted.

147. Insulin derivative according to any of paragraphs 143-146, wherein the amino acid residue in position A21 of the parent insulin is Gly or Asn.

148. Insulin derivative according to any of paragraphs 143-147, wherein the amino acid residue at position B3 of the parent insulin is Lys.

149. Insulin derivative according to any of paragraphs 143-148, wherein the amino acid residue at position B28 of the parent insulin is Asp or Lys.

150. Insulin derivative according to any of paragraphs 143-149, wherein the amino acid residue at position B29 of the parent insulin is Pro or Thr.

151. Insulin derivative according to paragraph 149, wherein the parent insulin is AspB28 human insulin.

152. Insulin derivative according to paragraph 147, wherein the parent insulin is GlyA21 human insulin or GlyA21desB30 human insulin or GlyA21ArgB31ArgB32 human insulin.

153. Insulin derivative according to paragraph 148, wherein the parent insulin is LysB3GluB29 human insulin.

154. Insulin derivative according to paragraph 149-150, wherein the parent insulin is LysB28ProB29 human insulin.

155. Insulin derivative according to paragraph 144 and 150, wherein the parent insulin is ThrB29LysB30 human insulin.

156. A zinc complex of an insulin derivative according to any one of the preceding paragraphs wherein two zinc ions, three zinc ions, four zinc ions, five zinc ions, six zinc ions, seven zinc ions, eight zinc ions, nine zinc ions or ten six zinc ions are bound per six molecules of insulin derivative.

157. A pharmaceutical composition for the treatment of diabetes in a patient in need of such treatment, comprising a therapeutically effective amount of an insulin derivative according to paragraphs 1-155 or a zinc complex according to paragraph 156 together with a pharmaceutically acceptable carrier.

158. A pharmaceutical composition for the treatment of diabetes in a patient in need of such treatment, comprising a therapeutically effective amount of an insulin derivative according to paragraphs 1-155 or a zinc complex according to paragraph 156 in mixture with an insulin or an insulin analogue which has a rapid onset of action, together with a pharmaceutically acceptable carrier.

159. A method of treating diabetes in a patient in need of such a treatment, comprising administering to the patient a therapeutically effective amount of an insulin derivative according to paragraphs 1-155 or a zinc complex according to paragraph 156 together with a pharmaceutically acceptable carrier.

160. A method of treating diabetes in a patient in need of such a treatment, comprising administering to the patient a therapeutically effective amount of an insulin derivative according to paragraphs 1-155 or a zinc complex according to paragraph 156 in mixture with an insulin or an insulin analogue which has a rapid onset of action, together with a pharmaceutically acceptable carrier.

161. A method according to paragraphs 138 or 139 for pulmonary treatment of diabetes.

162. Use of an insulin derivative according to paragraphs 1-155 or a zinc complex according to paragraph 156 for the manufacture of a pharmaceutical composition for the use in the treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia.

163. Use of an insulin derivative according to paragraphs 1-155 or a zinc complex according to paragraph 156 in mixture with an insulin or an insulin analogue which has a rapid onset of action for the manufacture of a pharmaceutical composition for the use in the treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia.

164. A mixture of an insulin derivative according to paragraphs 1-155 or a zinc complex according to paragraph 156 and a rapid acting insulin analogue selected group consisting of AspB28 human insulin; LysB28ProB29 human insulin and LysB3GluB29 human insulin.

165. An insulin derivative, wherein the insulin derivative is selected from the group consisting of:

-   N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-glutamylamide desB30 human     insulin, -   N^(εB29)-ω-carboxy-pentadecanoyl-γ-amino-butanoyl desB30 human     insulin, -   N^(εB29)-ω-carboxy-tetradecanoyl-γ-L-glutamylamide desB30 human     insulin, -   N^(εB29)-ω-carboxy-tridecanoyl-γ-L-glutamylamide desB30 human     insulin, -   N^(εB29)-ω-carboxy-pentadecanoyl-β-alanyl desB30 human insulin, -   N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-aspartylamide desB30 human     insulin, -   N^(εB29)-ω-carboxy-pentadecanoyl-ε-aminohexanoyl desB30 human     insulin, -   N^(εB29)-ω-carboxy-pentadecanoyl-δ-aminopentanoyl desB30 human     insulin, -   N^(εB29)-10-(4-carboxyphenoxy)-decanoyl-γ-L-glutamylamide desB30     human insulin, -   N^(εB29)-4-[11-(4-Carboxyphenyl)undecanoylamino]butyryl desB30 human     insulin, -   N^(εB29)-(3-(3-{4-[3-(7-carboxyheptanoylamino)propoxy]butoxy}propylcarbamoyl)-propionyl-γ-glutamylamide)     desB30 human Insulin, -   N^(εB29)-ω-carboxy-tridecanoyl-γ-amino-butanoyl desB30 human     insulin, -   N^(εB29)-ω-carboxy-undecanoyl-γ-amino-butanoyl desB30 human insulin, -   N^(εB29)-ω-carboxy-tetradecanoyl-γ-amino-butanoyl desB30 human     insulin, -   N^(εB29)-{4-[10-(4-Carboxy-phenoxy)-decanoylamino]-butyryl} desB30     insulin, -   N^(εB29)-{4-[(14-Carboxy-tetradecanoylamino)-methyl]-benzoyl} desB30     insulin, -   N^(εB29)-[16-(4-Carboxy-phenoxy)-hexadecanoyl] desB30 insulin, -   N^(εB29)-{4-[(15-carboxypentadecanoylamino)benzoyl]-desB30 human     insulin and -   N^(εB29)-{4-[(15-Carboxy-pentadecanoylamino)-methyl]-benzoyl}-desB30     insulin

166. Insulin derivative as described in the examples.

The invention will further be summarized in the following paragraphs:

1a. Insulin derivative having a substituent attached to a parent insulin moiety at either the α-amino group of the N-terminal amino acid residue of the B chain or at an ε-amino group of a Lys residue present in the B chain of the parent insulin moiety, which substituent comprises at least one fatty difunctionalized moiety with about 6 to about 32 carbon atoms and an uncharged linker which link the fatty difunctionalized moiety to the parent insulin, provided that the uncharged linker does not comprise —(CO—(CH₂)₂₋₆—NH—CO)₁₋₄— if the insulin substituent comprises an aromatic group.

2a. Insulin derivative according to paragraphs 1a, wherein the substituent is attached to the α-amino group of the N-terminal amino acid residue of the B-chain of the parent insulin.

3a. Insulin derivative according to paragraphs 1a, wherein the substituent is attached to the ε-amino group of a Lys residue present in the B-chain of the parent insulin.

4a. Insulin derivative according to paragraphs 1a or 3a wherein the substituent is attached to the ε-amino group of the Lys residue in position B29 present in the B-chain of the parent insulin.

5a. Insulin derivative according to paragraphs 4a, wherein the substituent is attached to the ε-amino group of the Lys residue in position B29 in LysB29desB30 human insulin

6a. Insulin derivative according to any of paragraphs 1a-5a, wherein the linker comprises an amide or a N-substituted amide of the formula —CONR₇R₈, where R₇ and R₈, independently of each other can be hydrogen, methyl, ethyl, propyl or isopropyl.

7a. Insulin derivative according to paragraphs 6a, where R₇ and R₈ are hydrogen.

8a. Insulin derivative according to paragraphs 1, wherein the linker comprises an amide.

9a. Insulin derivative according to any of paragraphs 1a-7a, wherein the substituent comprises one or more residues of ethyleneglycol, propyleneglycol and/or butyleneglycol containing independently at each termini a group selected from —NH₂ and —COOH.

10a. Insulin derivative according to paragraphs 1a-7a, wherein the substituent comprises at least one aromatic group.

11a. Insulin derivatives according to paragraphs 1a having the formula

wherein Ins is the parent insulin moiety which via the α-amino group of the N-terminal amino acid residue of the B chain or an ε-amino group of a Lys residue present in the B chain of the insulin moiety is bound to is bound to Q₁ or Q₂ in the substituent; each n is independently 0, 1, 2, 3, 4, 5 or 6; Q₁ is:

-   -   an α-amino acid amide residue having a carboxylic acid group in         the substituent which residue forms, with one of its carboxylic         acid groups, an amide group together with the α-amino group of         the N-terminal amino acid residue of the B chain or together         with the ε-amino group of a Lys residue present in the B chain         of the parent insulin;     -   a chain composed of two, three or four α-amino acid amide         residues linked together via amide bonds, which chain—via an         amide bond—is linked to the α-amino group of the N-terminal         amino acid residue of the B chain or to the ε-amino group of a         Lys residue present in the B chain of the parent insulin, the         amino acid residues of W being selected from the group of amino         acid residues having a neutral substituent and amino acid         residues having a carboxylic acid group in the substituent so         that W has at least one amino acid residue which has a         carboxylic acid group in the substituent; or     -   a bond         Q₂ is:

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—(CO—(CH₂)₂₋₆—NH—CO)₁₋₄—;

—(CO—(CH₂)₂₋₆—CO—NH)₁₋₄—;

—(CO—(CR₉R₁₀)₁₋₆—CO—NH)₁₋₄—, where R₉ and R₁₀, independently of each other can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂; or

a bond

-   -   provided that at least one of Q₁ or Q₂ is not a bond;         Q₃, Q₄, and Q₅ independently of each other can be     -   —(CH₂)_(m)— where m is an integer in the range of 1 to 32;     -   a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups         and a number of —CH₂— groups sufficient to give a total number         of carbon atoms in the chain in the range of 4 to 32;     -   (CH₂CH₂O)_(y)—; (CH₂CH₂CH₂O)_(y)—; (CH₂CH₂CH₂CH₂O)_(y)—;         (CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)— or (CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)—;         —(CH₂OCH₂)_(y)— where y is 1-20;     -   —(CR₃R₄)₁₋₆—(NHCO—(CR₃R₄)₁₋₆—NHCO)₁₋₂—(CR₃R₄)₁₋₆ or         —(CR₃R₄)₁₋₆—(CONH—(CR₃R₄)₁₋₆—CONH)₁₋₂—(CR₃R₄)₁₋₆—,         —(CR₃R₄)₁₋₆—(NHCO—(CR₃R₄)₁₋₆—CONH)₁₋₂—(CR₃R₄)₁₋₆— or         —(CR₃R₄)₁₋₆—(CONH—(CR₃R₄)₁₋₆—NHCO)₁₋₂—(CR₃R₄)₁₋₆ where R₃ and R₄         independently of each other and independently for each carbon         can be H, —COOH or OH,     -   —(CR₅R₆)₁₋₆—, where R₅ and R₆ independently of each other and         independently for each carbon can be H, —COOH, (CH₂)₁₋₆COOH;     -   —((CR₁R₂)₁₋₆—NR₁₅—CO)₁₋₄—, where R₁, R₂ and R₁₅ independently of         each other can be H, —CH₃, —CH₁₋₆CH₃, —SO₃H, —(CH₂)₁₋₆—SO₃H,         —(CH₂)₁₋₆—O—PO₃H₂ or CONH₂ and R₁₅ can be arylene which may be         substituted with one or two groups of R₁, R₂ as defined above;     -   NR₁₅ where R₁₅ is defined as above;     -   arylene or heteroarylene, which may be substituted with one or         two groups selected from the group consisting of —COOH, —CH₃,         —CH₁₋₆CH₃, —SO₃H, —(CH₂)_(p)—SO₃H, —CONR₁R₂ or —SO₂NR₁R₂, where         R₁ and R₂, independently of each other can be H, —CH₃,         —(CH)₁₋₆—CH₃, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂ or CONH₂;     -   a divalent hydrocarbon chain of the formula         —(CH₂)_(s)—Y₁—(C₆H₄)_(v1)—Y₂—(CH₂)_(w)—Y₃—(C₆H₄)_(v2)—Y₄—(CH₂)_(t)—Y₅—(C₆H₄)_(v3)—Y₆—(CH₂)_(z)—         wherein Y₁-Y₆ independently of each other can be O; S or a bond;         where s, w, t and z independently of each other are zero or an         integer from 1 to 10 so that the sum of s, w, t and z is in the         range from 4 to 30, and v₁, v₂, and v₃ independently of each         other can be zero or 1 with the proviso that Y₁-Y₆ do not link         to each other; or     -   a bond;         -   with the proviso that Q₃-Q₅ are different;             X₁, X₂ and X₃ are independently of each other:

O;

—C═O

a bond; or

-   -   where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;         and         Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)₂;

—N(CH₂COOH)₂;

—SO₃H

—OSO₃H

—OPO3H₂

—PO₃H₂ or

-tetrazol-5-yl or

—O—W₁,

-   -   where W₁ is arylene or heteroarylene, which may be substituted         with one or two groups selected from the group consisting of         —COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —CONR₁₃R₁₄ or —SO₂NR₁₂R₁₄, where         R₁₃ and R₁₄, independently of each other can be H, —SO₃H,         —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONH₂ or tetrazo-5-lyl; and         any Zn²⁺ complex thereof.

12a. Insulin derivative according to paragraphs 11a, wherein Q₁ is an amino acid amide residue having from 4 to 10 carbon atoms.

13a. Insulin derivative according to any of paragraphs 11a-12a, wherein Q₁ is selected from the group consisting of β-D-Asp-amide, β-L-Asp-amide, γ-L-Glu-amide and γ-D-Glu-amide.

14a. Insulin derivative according to paragraphs 11a, wherein Q₁ is a chain of amino acid amide residues.

15a. Insulin derivative according to paragraphs 11a or 14a, wherein Q₁ is a chain of two amino acid amide residues selected from the group consisting of β-L-Asp-amide-β-L-Asp-amide, β-L-Asp-amide-γ-L-Glu-amide, γ-L-Glu-amide-γ-L-Glu-amide, γ-L-Glu-amide-β-L-Asp-amide, β-L-Asp-amide-β-D-Asp-amide, β-L-Asp-amide-γ-D-Glu-amide, γ-L-Glu-amide-γ-D-Glu-amide, γ-L-Glu-amide-β-D-Asp-amide, β-D-Asp-amide-β-L-Asp-amide, β-D-Asp-amide-γ-L-Glu-amide, γ-D-Glu-amide-γ-L-Glu-amide, γ-D-Glu-amide-β-L-Asp-amide, β-D-Asp-amide-β-D-Asp-amide, β-D-Asp-amide-γ-D-Glu-amide, γ-D-Glu-amide-γ-D-Glu-amide, γ-D-Glu-amide-β-D-Asp-amide,

16a. Insulin derivative according to any of paragraphs 11a-15a, wherein Q₂ is a bond

17a. Insulin derivative according to any of paragraphs 11a-15a, wherein Q₂ is selected from the group consisting of —(CO—(CH₂)₂₋₆—NH—CO)₁₋₄—; —(CO—(CH₂)₂₋₆—CO—NH)₁₋₄—;

18a. Insulin derivative according to paragraphs 17a, wherein Q₂ is selected from the group consisting of —(CO—(CH₂)₂—NH—CO)₁— or —(CO—(CH₂)₃—NH—CO)₁₋₄.

19a. Insulin derivative according to paragraphs 17a-18a, wherein Q₁ is a bond.

20a. Insulin derivative according to paragraphs 11a wherein

Q₁ is:

-   -   an α-amino acid amide residue having a carboxylic acid group in         the substituent which residue forms, with one of its carboxylic         acid groups, an amide group together with the α-amino group of         the N-terminal amino acid residue of the B chain or together         with the ε-amino group of a Lys residue present in the B chain         of the parent insulin;     -   a chain composed of two, three or four α-amino acid amide         residues linked together via amide bonds, which chain—via an         amide bond—is linked to the α-amino group of the N-terminal         amino acid residue of the B chain or to the ε-amino group of a         Lys residue present in the B chain of the parent insulin, the         amino acid residues of W being selected from the group of amino         acid residues having a neutral substituent and amino acid         residues having a carboxylic acid group in the substituent so         that W has at least one amino acid residue which has a         carboxylic acid group in the substituent; or     -   a bond         Q₂ is:

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—(CO—(CH₂)₂₋₆—NH—CO)₁₋₄—;

—(CO—(CH₂)₂₋₆—CO—NH)₁₋₄—;

—(CO—(CR₉R₁₀)₁₋₆—CO—NH)₁₋₄—, where R₉ and R₁₀, independently of each other can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂; or

a bond

-   -   provided that at least one of Q₁ or Q₂ is not a bond;     -   Q₃ is     -   —(CH₂)_(m)— where m is an integer in the range of 6 to 32;     -   a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups         and a number of —CH₂— groups sufficient to give a total number         of carbon atoms in the chain in the range of 4 to 32; or     -   a divalent hydrocarbon chain of the formula         —(CH₂)_(s)C₆H₄(CH₂)_(w)— wherein v and w are integers or one of         them is zero so that the sum of s and w is in the range of 6 to         30;         -   X₁, can be —C═O or a bond;         -   Q₄, Q₅, X₂ and X₃ are bonds;             All values of n are zero; and             Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)₂;

—N(CH₂COOH)₂;

—SO₃H

—OSO₃H

—OPO3H₂

—PO₃H₂ or

-tetrazol-5-yl

and any Zn²⁺ complex thereof

21a. Insulin derivative according to paragraphs 20a, wherein Q₁ is an amino acid amide residue having from 4 to 10 carbon atoms.

22a. Insulin derivative according to any of paragraphs 20a-21a, wherein Q₁ is selected from the group consisting of β-D-Asp-amide, β-L-Asp-amide, γ-L-Glu-amide and γ-D-Glu-amide.

23a. Insulin derivative according to paragraphs 20a, wherein Q₁ is a chain of amino acid amide residues.

24a. Insulin derivative according to paragraphs 20a or 23a, wherein Q₁ is a chain of two amino acid amide residues selected from the group consisting of β-L-Asp-amide-β-L-Asp-amide, β-L-Asp-amide-γ-L-Glu-amide, γ-L-Glu-amide-γ-L-Glu-amide, γ-L-Glu-amide-β-L-Asp-amide, β-L-Asp-amide-β-D-Asp-amide, β-L-Asp-amide-γ-D-Glu-amide, γ-L-Glu-amide-γ-D-Glu-amide, γ-L-Glu-amide-β-D-Asp-amide, β-D-Asp-amide-β-L-Asp-amide, β-D-Asp-amide-γ-L-Glu-amide, γ-D-Glu-amide-γ-L-Glu-amide, γ-D-Glu-amide-β-L-Asp-amide, β-D-Asp-amide-β-D-Asp-amide, β-D-Asp-amide-γ-D-Glu-amide, γ-D-Glu-amide-γ-D-Glu-amide, γ-D-Glu-amide-β-D-Asp-amide,

25a. Insulin derivative according to any of paragraphs 20a-24a, wherein Q₂ is a bond

26a. Insulin derivative according to any of paragraphs 21a-25a, wherein X₁ is —C═O

27a. Insulin derivative according to any of paragraphs 20a-26a, wherein Q₂ is selected from the group consisting of CO(CONH₂)CH—; —(CO—(CH₂)₂₋₆—NH—CO)₁₋₄—; —(CO—(CH₂)₂₋₆—CO—NH)₁₋₄—;

28a. Insulin derivative according to any of paragraphs 20a or 27a, wherein Q₂ is selected from the group consisting of —(CO—(CH₂)₂—NH—CO)₁—-(CO—(CH₂)₃—NH—CO)₁—, —(CO—(CH₂)₄—NH—CO)₁— or —(CO—(CH₂)₅—NH—CO)₁—.

29a. Insulin derivative according to paragraphs 27a or 28a, wherein Q₁ is a bond.

30a. Insulin derivative according to any of paragraphs 20a-29a, wherein Q₃ is —(CH₂)_(m)— where m is an integer in the range of 6 to 32 or from 8 to 20.

31a. Insulin derivative according to paragraphs 30a, where m is 12, 13, 14, 15 or 16.

32a. Insulin derivative according to any of paragraphs 20a-31a, wherein Z is —COOH.

33a. Insulin derivative according to any of paragraphs 20a-31a, wherein Z is —CH(COOH)₂.

34a. An insulin derivative according to any of paragraphs 20a-31a, wherein Z is —N(CH₂COOH)₂.

35a. An insulin derivative according to any of paragraphs 20a-31a, wherein Z is —SO₃H.

36a. An insulin derivative according to any of paragraphs 20a-31a, wherein Z is —PO₃H.

37a. Insulin derivative according to any of the preceeding paragraphs selected from the group consisting of N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-glutamylamide desB30 human insulin, N^(εB29)-ω-carboxy-pentadecanoyl-γ-amino-butanoyl desB30 human insulin, N^(εB29)-ω-carboxy-tetradecanoyl-γ-L-glutamylamide desB30 human insulin, N^(εB29)-ω-carboxy-tridecanoyl-γ-L-glutamylamide desB30 human insulin, N^(εB29)-ω-carboxy-pentadecanoyl-β-alanyl desB30 human insulin, N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-aspartylamide desB30 human insulin, N^(εB29)-ω-carboxy-pentadecanoyl-ε-aminohexanoyl desB30 human insulin, N^(εB29)-ω-carboxy-pentadecanoyl-δ-aminopentanoyl desB30 human insulin.

38a. Insulin derivative according to paragraphs 11a, wherein

Q₁ is:

-   -   an α-amino acid amide residue having a carboxylic acid group in         the substituent which residue forms, with one of its carboxylic         acid groups, an amide group together with the α-amino group of         the N-terminal amino acid residue of the B chain or together         with the ε-amino group of a Lys residue present in the B chain         of the parent insulin;     -   a chain composed of two, three or four α-amino acid amide         residues linked together via amide bonds, which chain—via an         amide bond—is linked to the α-amino group of the N-terminal         amino acid residue of the B chain or to the ε-amino group of a         Lys residue present in the B chain of the parent insulin, the         amino acid residues of W being selected from the group of amino         acid residues having a neutral substituent and amino acid         residues having a carboxylic acid group in the substituent so         that W has at least one amino acid residue which has a         carboxylic acid group in the substituent; or     -   a bond         Q₂ is:

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—(CO—(CH₂)₂₋₆—NH—CO)₁₋₄—;

—(CO—(CH₂)₂₋₆—CO—NH)₁₋₄—;

—(CO—(CR₉R₁₀)₁₋₆—CO—NH)₁₋₄—, where R₉ and R₁₀, independently of each other can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂; or

a bond

-   -   provided that at least one of Q₁ or Q₂ is not a bond;     -   Q₃ is     -   —((CR₁R₂)₁₋₆—NR₁₅—CO)₁₋₄—, where R₁, R₂ and R₁₅ independently of         each other can be H, —CH₃, —CH₁₋₆CH₃, —SO₃H, —(CH₂)₁₋₆—SO₃H,         —(CH₂)₁₋₆—O—PO₃H₂ or CONH₂ and R₁₅ can be arylene which may be         substituted with one or two groups of R₁, R₂ as defined above;     -   NR₁₅ where R₁₅ is defined as above,     -   a bond         Q₄ is     -   —(CH₂)_(m)— where m is an integer from 4 to 22;     -   a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups         and a number of —CH₂— groups sufficient to give a total number         of carbon atoms in the chain in the range of 4 to 22;     -   arylene or heteroarylene, which may be substituted with one or         two groups selected from the group consisting of —COOH, —CH₃,         —CH_(p)CH₃, —SO₃H, —(CH₂)_(p)—SO₃H, —CONR₁R₂ or —SO₂NR₁R₂, where         R₁ and R₂, independently of each other can be H, —CH₃,         —(CH)₁₋₆—CH₃, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)_(p)—O—PO₃H₂ or         CONH₂; or     -   a divalent hydrocarbon chain of the formula         —(CH₂)_(s)—Y₁—(C₆H₄)_(v1)—Y₂—(CH₂)_(w)—Y₃—(C₆H₄)_(v2)—Y₄—(CH₂)_(t)—Y₅—(C₆H₄)_(v3)—Y₆—(CH₂)_(z)—     -   wherein Y₁-Y₆ independently of each other can be O; S or a bond;         where s, w, t and z independently of each other are zero or an         integer from 1 to 10 so that the sum of s, w, t and z is in the         range from 4 to 30, and v₁, v₂, and v₃ independently of each         other can be zero or 1 with the proviso that Y₁-Y₆ do not link         to each other; or X₁ is

O;

—C═O

a bond; or

-   -   where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;         and X₂, X₃ and Q₅ are bonds;         All values of n are zero; and

Z is:

-   -   —COOH;     -   —CO-Asp;     -   —CO-Glu;     -   —CO-Gly;     -   —CO-Sar;     -   —CH(COOH)₂;     -   —N(CH₂COOH)₂;     -   —SO₃H     -   —OSO₃H     -   —OPO3H₂     -   —PO₃H₂ or     -   -tetrazol-5-yl or     -   —O—W₁,     -   where W₁ is arylene or heteroarylene, which may be substituted         with one or two groups selected from the group consisting of         —COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —CONR₁₃R₁₄ or —SO₂NR₁₂R₁₄, where         R₁₃ and R₁₄, independently of each other can be H, —SO₃H,         —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONH₂ or tetrazo-5-lyl;         and any Zn²⁺ complex thereof.

39a. Insulin derivative according to paragraphs 38a, wherein Q₁ is an amino acid amide residue having from 4 to 10 carbon atoms.

40a. Insulin derivative according to any of paragraphs 38a-39a, wherein Q₁ is selected from the group consisting of β-D-Asp-amide, β-L-Asp-amide, γ-L-Glu-amide and γ-D-Glu-amide.

41a. Insulin derivative according to paragraphs 38a, wherein Q₁ is a chain of amino acid amide residues.

42a. Insulin derivative according to paragraphs 38a or 41a, wherein Q₁ is a chain of two amino acid amide residues selected from the group consisting of β-L-Asp-amide-β-L-Asp-amide, β-L-Asp-amide-γ-L-Glu-amide, γ-L-Glu-amide-γ-L-Glu-amide, γ-L-Glu-amide-β-L-Asp-amide, β-L-Asp-amide-β-D-Asp-amide, β-L-Asp-amide-γ-D-Glu-amide, γ-L-Glu-amide-γ-D-Glu-amide, γ-L-Glu-amide-β-D-Asp-amide, β-D-Asp-amide-β-L-Asp-amide, β-D-Asp-amide-γ-L-Glu-amide, γ-D-Glu-amide-γ-L-Glu-amide, γ-D-Glu-amide-β-L-Asp-amide, β-D-Asp-amide-β-D-Asp-amide, β-D-Asp-amide-γ-D-Glu-amide, γ-D-Glu-amide-γ-D-Glu-amide, γ-D-Glu-amide-β-D-Asp-amide,

43a Insulin derivative according to any of paragraphs 38a-42a, wherein Q₂ is a bond

44a. Insulin derivative according to any of paragraphs 38a-42a, wherein Q₂ is selected from the group consisting of CO(CONH₂)CH—; —(CO—(CH₂)₂₋₆—NH—CO)₁₋₄— and —(CO—(CH₂)₂₋₆—CO—NH)₁₋₄—.

45a. Insulin derivative according to paragraphs 44a, wherein Q₁ is a bond.

46a. Insulin derivative according to any of paragraphs 38a, 44a or 45a, wherein Q₂ is selected from the group consisting of —(CO—(CH₂)₂—NH—CO)₁— or —(CO—(CH₂)₃—NH—CO)₁₋₄—

47a. Insulin derivative according to paragraphs 37a, wherein Q₄ is —(CH₂)_(s)—Y₁—(C₆H₄)_(v1)—Y₂—(CH₂)_(w)—Y₃—(C₆H₄)_(v2)—Y₄—(CH₂)_(t)—Y₅—(C₆H₄)_(v3)—Y₆—(CH₂)_(z)—

wherein Y₁-Y₆ independently of each other can be O; S or a bond; where s, w, t and z independently of each other are zero or an integer from 1 to 10 so that the sum of s, w, t and z is in the range from 4 to 30, and v₁, v₂, and v₃ independently of each other can be zero or 1 with the proviso that Y₁-Y₆ do not link to each other.

48a. Insulin derivative according to any of paragraphs 37a or 46a, wherein at least two of v₁, v₂, or v₃ are zero.

49a. Insulin derivative according to any of paragraphs 38a, 47a or 48a, wherein at Y₁—Y₆ are bonds.

50a. Insulin derivative according to any of paragraphs 38a, 47a or 48a, wherein at least one of Y₁-Y₆ are O or S.

51a. Insulin derivative according to any of paragraphs 38a or 47a-48a, wherein Y₁ is O or S and v₁ is one.

52a. Insulin derivative according to any of paragraphs 38a-51a, wherein Z is —COOH.

53a. Insulin derivative according to any of paragraphs 38a-51a, wherein Z is —CH(COOH)₂.

54a. An insulin derivative according to any of paragraphs 38a-51a, wherein Z is —N(CH₂COOH)₂.

55a. An insulin derivative according to any of paragraphs 38a-51a, wherein Z is —SO₃H.

56a. An insulin derivative according to any of paragraphs 38a-51a, wherein Z is —PO₃H.

57a. An insulin derivative according to any of paragraphs 38a-51a, wherein Z is —O—W₁,

where W₁ is arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —COOH, —SO₃H, —(CH₂)₁₋₆—SO₃H, —CONR₁₃R₁₄ or —SO₂NR₁₂R₁₄, where R₁₃ and R₁₄, independently of each other can be H, —SO₃H, —(CH₂)₁₋₆—SO₃H, —(CH₂)₁₋₆—O—PO₃H₂, —CONH₂ or tetrazo-5-lyl.

58a. Insulin derivative according to any of paragraphs 1a-19a or 38a-57a wherein the insulin derivative are selected from the group consisting of N^(εB29)-10-(4-carboxyphenoxy)-decanoyl-γ-L-glutamylamide desB30 human insulin, N^(εB29)-4-[11-(4-Carboxyphenyl)undecanoylamino]butyryl desB30 human insulin.

59a. Insulin derivative according to paragraphs 11a, wherein

Q₁ is:

-   -   an α-amino acid amide residue having a carboxylic acid group in         the substituent which residue forms, with one of its carboxylic         acid groups, an amide group together with the α-amino group of         the N-terminal amino acid residue of the B chain or together         with the ε-amino group of a Lys residue present in the B chain         of the parent insulin;     -   a chain composed of two, three or four α-amino acid amide         residues linked together via amide bonds, which chain—via an         amide bond—is linked to the α-amino group of the N-terminal         amino acid residue of the B chain or to the ε-amino group of a         Lys residue present in the B chain of the parent insulin, the         amino acid residues of W being selected from the group of amino         acid residues having a neutral substituent and amino acid         residues having a carboxylic acid group in the substituent so         that W has at least one amino acid residue which has a         carboxylic acid group in the substituent; or     -   a bond         Q₂ is:

—COCH(CONH₂)—

—COCH₂N(CH₂CONH₂)—

—COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂)

—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)—

—COCH₂N(CH₂CH₂CONH₂)—

—COCH₂CH₂N(CH₂CONH₂)—

—(CO—(CH₂)₂₋₆—NH—CO)₁₋₄—;

—(CO—(CH₂)₂₋₆—CO—NH)₁₋₄—;

—(CO—(CR₉R₁₀)₁₋₆—CO—NH)₁₋₄—, where R₉ and R₁₀, independently of each other can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂; or

a bond

-   -   provided that at least one of Q₁ or Q₂ is not a bond;         n is independently 2 or 3;         Q₃, Q₄, and Q₅ independently of each other can be     -   (CH₂CH₂O)_(y)—; (CH₂CH₂CH₂O)_(y)—; (CH₂CH₂CH₂CH₂O)_(y)—;         (CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)— or (CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)—;         —(CH₂OCH₂)_(y)— where y is 1-20;     -   —(CH₂)_(m)— where m is an integer in the range of 1 to 32;     -   a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups         and a number of —CH₂— groups sufficient to give a total number         of carbon atoms in the chain in the range of 4 to 32;     -   —(CR₃R₄)₁₋₆—(NHCO—(CR₃R₄)₁₋₆—NHCO)₁₋₂—(CR₃R₄)₁₋₆ or         —(CR₃R₄)₁₋₆—(CONH—(CR₃R₄)₁₋₆—CONH)₁₋₂—(CR₃R₄)₁₋₆—,         —(CR₃R₄)₁₋₆—(NHCO—(CR₃R₄)₁₋₆—CONH)₁₋₂—(CR₃R₄)₁₋₆— or         —(CR₃R₄)₁₋₆—(CONH—(CR₃R₄)₁₋₆—NHCO)₁₋₂—(CR₃R₄)₁₋₆ where R₃ and R₄         independently of each other and independently for each carbon         can be H, —COOH or OH,     -   —(CR₅R₆)₁₋₆—, where R₅ and R₆ independently of each other and         independently for each carbon can be H, —COOH, (CH₂)₁₋₆COOH; or     -   a bond;         -   with the proviso that Q₃-Q₅ are different;             X₁, X₂ and X₃ are independently

O;

—C═O;

a bond; or

-   -   where R is hydrogen, C₁₋₃-alkyl, C₂₋₃-alkenyl or C₂₋₃-alkynyl;         and     -   Z is:     -   —COOH;     -   —CO-Asp;     -   —CO-Glu;     -   —CO-Gly;     -   —CO-Sar;     -   —CH(COOH)₂;     -   —N(CH₂COOH)₂;     -   —SO₃H     -   —OSO₃H     -   —OPO3H₂     -   —PO₃H₂ or     -   -tetrazol-5-yl         and any Zn²⁺ complex thereof.

60a. Insulin derivative according to paragraphs 59a, wherein Q₁ is an amino acid amide residue having from 4 to 10 carbon atoms.

61a. Insulin derivative according to any of paragraphs 59a-60a, wherein Q₁ is selected from the group consisting of β-D-Asp-amide, β-L-Asp-amide, γ-L-Glu-amide and γ-D-Glu-amide.

62a. Insulin derivative according to paragraphs 59a, wherein Q₁ is a chain of amino acid amide residues.

63a. Insulin derivative according to paragraphs 59a or 62a, wherein Q₁ is a chain of two amino acid amide residues selected from the group consisting of β-L-Asp-amide-β-L-Asp-amide, β-L-Asp-amide-γ-L-Glu-amide, γ-L-Glu-amide-γ-L-Glu-amide, γ-L-Glu-amide-β-L-Asp-amide, β-L-Asp-amide-β-D-Asp-amide, β-L-Asp-amide-γ-D-Glu-amide, γ-L-Glu-amide-γ-D-Glu-amide, γ-L-Glu-amide-β-D-Asp-amide, β-D-Asp-amide-β-L-Asp-amide, β-D-Asp-amide-γ-L-Glu-amide, γ-D-Glu-amide-γ-L-Glu-amide, γ-D-Glu-amide-β-L-Asp-amide, β-D-Asp-amide-β-D-Asp-amide, β-D-Asp-amide-γ-D-Glu-amide-γ-D-Glu-amide-γ-D-Glu-amide, γ-D-Glu-amide-β-D-Asp-amide,

64a. Insulin derivative according to any of paragraphs 59a-63a, wherein Q₂ is a bond

65a. Insulin derivative according to any of paragraphs 59a-63a, wherein Q₂ is selected from the group consisting of CO(CONH₂)CH—; —(CO—(CH₂)₂₋₆—NH—CO)₁₋₄—; —(CO—(CH₂)₂₋₆—CO—NH)₁₋₄—;

66a. Insulin derivative according to any of paragraphs 59a or 65a, wherein Q₂ is selected from the group consisting of —(CO—(CH₂)₂—NH—CO)₁— or —(CO—(CH₂)₃—NH—CO)₁₋₄—

67a. Insulin derivative according to paragraphs 65a or 66a, wherein Q₁ is a bond.

68a. Insulin derivative according any of paragraphs 59a-67a, wherein one of Q₃, Q₄, or Q₅ is —(CH₂)_(m)— where m is an integer in the range of 1 to 32 or 1-12.

69a. Insulin derivative according to any of paragraphs 59a-67a, wherein one of Q₃, Q₄, or Q₅ is (CH₂CH₂O)_(y)—; (CH₂CH₂CH₂O)_(y)—; (CH₂CH₂CH₂CH₂O)_(y)—; (CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)— or (CH₂CH₂CH₂OCH₂CH₂CH₂CH₂O)_(y)—; —(CH₂OCH₂)_(y)— where y is 1-20;

70a. Insulin derivative according to paragraphs 69a, wherein one of Q₃, Q₄, or Q₅ is (CH₂CH₂O)_(y)— or (CH₂CH₂OCH₂CH₂CH₂CH₂O) wherein y is in the range of 2-12, 2-4 or 2-3

71a. Insulin derivative according to any of paragraphs 59a, 69a or 70a, wherein y is 1.

72a. Insulin derivative according to any of paragraphs 59a-71a, wherein Z is —COOH.

73a. Insulin derivative according to any of paragraphs 59a-71a, wherein Z is —CH(COOH)₂.

74a. An insulin derivative according to any of paragraphs 59a-71a, wherein Z is —N(CH₂COOH)₂.

75a. An insulin derivative according to any of paragraphs 59a-71a, wherein Z is —SO₃H.

76a. An insulin derivative according to any of paragraphs 59a-71a, wherein Z is —PO₃H.

77a. Insulin derivative according to any of paragraphs 1a-19a or 59a-76a selected from the group consisting of

-   N^(εB29)-(3-(3-{4-[3-(7-carboxyheptanoylamino)propoxy]butoxy}propylcarbamoyl)-propionyl-γ-glutamylamide)     desB30 human Insulin

78a. Insulin derivative according to any of the paragraphs 1a-77a, wherein the parent insulin is human insulin or porcine insulin

79a Insulin derivative according to any of the paragraphs 1a77a, wherein the parent insulin is an insulin analogue.

80a. Insulin derivative according to any of paragraphs 78a-79a, wherein the amino acid residue at position B30 of the parent insulin is Lys or has been deleted.

81a. Insulin derivative according to paragraphs 80a, wherein the parent insulin is desB30 human insulin.

82a. Insulin derivative according to any of paragraphs 78a-81a, wherein the amino acid residue at position B1 of the parent insulin has been deleted.

83a. Insulin derivative according to any of paragraphs 78a-82a, wherein the amino acid residue in position A21 of the parent insulin is Gly or Asn.

84a. Insulin derivative according to any of paragraphs 78a-83a, wherein the amino acid residue at position B3 of the parent insulin is Lys

85a. Insulin derivative according to any of paragraphs 78a-84a, wherein the amino acid residue at position B28 of the parent insulin is Asp or Lys.

86a. Insulin derivative according to any of paragraphs 78a-85a, wherein the amino acid residue at position B29 of the parent insulin is Pro or Thr.

87a. Insulin derivative according to paragraphs 85a, wherein the parent insulin is AspB28 human insulin

88a. Insulin derivative according to paragraphs 83a, wherein the parent insulin is GlyA21 human insulin or GlyA21desB30 human insulin or GlyA21ArgB31ArgB32 human insulin.

89a. Insulin derivative according to paragraphs 84a, wherein the parent insulin is LysB3GluB29 human insulin.

90a. Insulin derivative according to paragraphs 85a-86a, wherein the parent insulin is LysB28ProB29 human insulin

91a. Insulin derivative according to paragraphs 80a and 86a, wherein the parent insulin is ThrB29LysB30 human insulin

92a. A zinc complex of an insulin derivative according to any of the preceding paragraphs wherein each insulin hexamer binds two zinc ions, three zinc ions or four zinc ions.

93a. A pharmaceutical composition for the treatment of diabetes in a patient in need of such treatment, comprising a therapeutically effective amount of an insulin derivative according to any of the preceding paragraphs together with a pharmaceutically acceptable carrier.

94a. A pharmaceutical composition for the treatment of diabetes in a patient in need of such treatment, comprising a therapeutically effective amount of an insulin derivative according to any of the preceding paragraphs in mixture with an insulin or an insulin analogue which has a rapid onset of action, together with a pharmaceutically acceptable carrier.

95a. A method of treating diabetes in a patient in need of such a treatment, comprising administering to the patient a therapeutically effective amount of an insulin derivative according to paragraphs 1a-94a together with a pharmaceutically acceptable carrier.

94a. A method of treating diabetes in a patient in need of such a treatment, comprising administering to the patient a therapeutically effective amount of an insulin derivative according to paragraphs 1a-94a in mixture with an insulin or an insulin analogue which has a rapid onset of action, together with a pharmaceutically acceptable carrier.

97a. A method according to paragraphs 95a or 96a for pulmonary treatment of diabetes

98a. A mixture of an insulin derivative according to any of paragraphs 1a-92a and a rapid acting insulin analogue selected group consisting of AspB28 human insulin; LysB28ProB29 human insulin and LysB3GluB29 human insulin.

99a. Insulin derivative as described in the examples.

The starting product for the acylation, the parent insulin or insulin analogue or a precursor thereof can be produced by either well-know peptide synthesis or by well known recombinant production in suitable transformed microorganisms. Thus the insulin starting product can be produced by a method which comprises culturing a host cell containing a DNA sequence encoding the polypeptide and capable of expressing the polypeptide in a suitable nutrient medium under conditions permitting the expression of the peptide, after which the resulting peptide is recovered from the culture.

The medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection). The peptide produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, gel filtration chromatography, affinity chromatography, or the like, dependent on the type of peptide in question.

The DNA sequence encoding the parent insulin may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the polypeptide by hybridisation using synthetic oligonucleotide probes in accordance with standard techniques (see, for example, Sambrook, J. Fritsch, E F and Maniatis, T. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989). The DNA sequence encoding the parent insulin may also be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801-805. The DNA sequence may also be prepared by polymerase chain reaction using specific primers, for instance as described in U.S. Pat. No. 4,683,202 or Saiki et al., Science 239 (1988), 487-491.

The DNA sequence may be inserted into any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

The vector is for example an expression vector in which the DNA sequence encoding the parent insulin is operably linked to additional segments required for transcription of the DNA, such as a promoter. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA encoding the parent insulin in a variety of host cells are well known in the art, cf. for instance Sambrook et al., supra.

The DNA sequence encoding the parent insulin may also, if necessary, be operably connected to a suitable terminator, polyadenylation signals, transcriptional enhancer sequences, and translational enhancer sequences. The recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.

The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell or one which confers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate.

To direct a peptide of the present invention into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector. The secretory signal sequence is joined to the DNA sequence encoding the peptide in the correct reading frame. Secretory signal sequences are commonly positioned 5′ to the DNA sequence encoding the peptide. The secretory signal sequence may be that normally associated with the peptide or may be from a gene encoding another secreted protein.

The procedures used to ligate the DNA sequences coding for the parent insulin, the promoter and optionally the terminator and/or secretory signal sequence, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., supra).

The host cell into which the DNA sequence or the recombinant vector is introduced may be any cell which is capable of producing the present peptide and includes bacteria, yeast, fungi and higher eukaryotic cells. Examples of suitable host cells well known and used in the art are, without limitation, E. coli, Saccharomyces cerevisiae, or mammalian BHK or CHO cell lines.

The parent insulin molecule is then converted into the insulin derivatives of the invention by introducing of the relevant substituent in either the B1 position or in the chosen Lys position in the B-chain. The substituent can be introduced by any convenient method and many methods are disclosed in the prior art for acylation of an amino group. More details will appear from the following examples.

Insulin derivatives according to the invention may be provided in the form of essentially zinc free compounds or in the form of zinc complexes. When zinc complexes of an insulin derivative according to the invention are provided, two Zn²⁺ ions, three Zn²⁺ ions, four Zn²⁺ ions, five Zn²⁺ ions, six Zn²⁺ ions, seven Zn²⁺ ions, eight Zn²⁺ ions, nine Zn²⁺ ions or ten Zn²⁺ ions can be bound per six molecules of insulin derivative. Solutions of zinc complexes of the insulin derivatives will contain mixtures of such species.

In one aspect the invention is related to a pharmaceutical composition comprising a therapeutically effective amount of an insulin derivative or a zinc complex of the insulin derivative according to the invention optionally together with a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable additive, which composition can be provided for the treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia in patients in need of such a treatment.

In one aspect of the invention, there is provided a method of treating type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia in a patient in need of such a treatment, comprising administering to the patient a therapeutically effective amount of an pharmaceutical composition comprising the insulin derivative or a zinc complex of the insulin derivative according to the invention optionally together with a pharmaceutically acceptable carrier and/or pharmaceutical acceptable additives.

In one aspect of the invention, there is provided a method for the manufacture of a pharmaceutical composition for the use in the treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia, the composition comprising an insulin derivative or a zinc complex of the insulin derivative according to the invention optionally together with a pharmaceutically acceptable carrier and/or pharmaceutical acceptable additives.

In one aspect of the invention, there is provided a pharmaceutical composition for treating type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia in a patient in need of such a treatment, the composition comprising a therapeutically effective amount of an insulin derivative or a zinc complex of the insulin derivative according to the invention in mixture with an insulin or an insulin analogue which has a rapid onset of action, optionally together with pharmaceutically acceptable carriers and/or additives.

In one aspect of the invention, there is provided a method of treating type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia in a patient in need of such a treatment, comprising administering to the patient a therapeutically effective amount of an pharmaceutical composition comprising the insulin derivative or a zinc complex of the insulin derivative according to the invention in mixture with an insulin or an insulin analogue which has a rapid onset of action, optionally together with a pharmaceutically acceptable carrier and/or pharmaceutical acceptable additives.

In one aspect of the invention, there is provided a method for the manufacture of a pharmaceutical composition for the use in the treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia, the composition comprising a therapeutically effective amount of an insulin derivative or a zinc complex of the insulin derivative according to the invention in mixture with an insulin or an insulin analogue which has a rapid onset of action, optionally together with a pharmaceutically acceptable carrier and/or pharmaceutical acceptable additives.

In one aspect the invention provides a pharmaceutical composition being a mixture of an insulin derivative or a zinc complex of the insulin derivative according to the invention and a rapid acting insulin analogue selected group consisting of AspB28 human insulin; LysB28ProB29 human insulin and LysB3GluB29 human insulin.

One aspect of the invention is related to a pharmaceutical composition comprising a therapeutically effective amount of an insulin derivative or a zinc complex of the insulin derivative according to the invention optionally together with a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable additive, which can be provided for pulmonary treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia in patients in need of such a treatment.

In one aspect the invention is related to application of a pharmaceutical composition for pulmonary treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia in a patient in need of such a treatment, the pharmaceutical composition comprising a therapeutically effective amount of an insulin derivative or a zinc complex of the insulin derivative according to the invention optionally in mixture with an insulin or an insulin analogue which has a rapid onset of action, and optionally together with pharmaceutically acceptable carriers and/or additives.

In one aspect of the invention, there is provided a method for the manufacture of a pharmaceutical composition for the use in the treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia, the composition being used pulmonary and comprising a therapeutically effective amount of an insulin derivative or a zinc complex of the insulin derivative according to the invention optionally in mixture with an insulin or an insulin analogue which has a rapid onset of action, and optionally together with a pharmaceutically acceptable carrier and/or pharmaceutical acceptable additives.

The insulin derivative according to the invention and the rapid acting insulin analogue can be mixed in a ratio from about 90/10%; about 70/30% or about 50/50%.

In one aspect, the invention relates to a pharmaceutical composition comprising an insulin derivative according to the invention which is soluble at physiological pH values.

In one aspect, the invention relates to a pharmaceutical composition comprising an insulin derivative according to the invention which is soluble at pH values in the interval from about 6.5 to about 8.5.

In one aspect, the invention relates to a pharmaceutical composition with a prolonged profile of action which comprises an insulin derivative according to the invention.

In one aspect, the invention relates to a pharmaceutical composition which is a solution containing from about 120 nmol/ml to about 2400 nmol/ml, from about 400 nmol/ml to about 2400 nmol/ml, from about 400 nmol/ml to about 1200 nmol/ml, from about 600 nmol/ml to about 2400 nmol/ml, or from about 600 nmol/ml to about 1200 nmol/ml of an insulin derivative according to the invention or of a mixture of the insulin derivative according to the invention with a rapid acting insulin analogue.

Pharmaceutical Compositions

The insulin derivatives of this invention of the claimed formula can, for example, be administered subcutaneously, orally, or pulmonary.

For subcutaneous administration, the compounds of the formula are formulated analogously with the formulation of known insulins. Furthermore, for subcutaneous administration, the compounds of the formula are administered analogously with the administration of known insulins and, generally, the physicians are familiar with this procedure.

The insulin derivatives of this invention may be administered by inhalation in a dose effective manner to increase circulating insulin levels and/or to lower circulating glucose levels. Such administration can be effective for treating disorders such as diabetes or hyperglycemia. Achieving effective doses of insulin requires administration of an inhaled dose of insulin derivative of this invention of more than about 0.5 μg/kg to about 50 μg/kg. A therapeutically effective amount can be determined by a knowledgeable practitioner, who will take into account factors including insulin level, blood glucose levels, the physical condition of the patient, the patient's pulmonary status, or the like.

Administration by inhalation can result in pharmacokinetics comparable to subcutaneous administration of insulins. Different inhalation devices typically provide similar pharmacokinetics when similar particle sizes and similar levels of lung deposition are compared.

According to the invention, insulin derivative of this invention may be delivered by any of a variety of inhalation devices known in the art for administration of a therapeutic agent by inhalation. These devices include metered dose inhalers, nebulizers, dry powder generators, sprayers, and the like. Insulin derivative of this invention is delivered by a dry powder inhaler or a sprayer. There are a several desirable features of an inhalation device for administering insulin derivative of this invention. For example, delivery by the inhalation device is advantageously reliable, reproducible, and accurate. The inhalation device should deliver small particles, for example, less than about 10 μm, for example about 1-5 μm, for good respirability. Some specific examples of commercially available inhalation devices suitable for the practice of this invention are Turbohaler™ (Astra), Rotahaler® (Glaxo), Diskus® (Glaxo), Spiros™ inhaler (Dura), devices marketed by Inhale Therapeutics, AERx™ (Aradigm), the Ultravent® nebulizer (Mallinckrodt), the Acorn II® nebulizer (Marquest Medical Products), the Ventolin® metered dose inhaler (Glaxo), the Spinhaler® powder inhaler (Fisons), or the like.

As those skilled in the art will recognize, the formulation of insulin derivative of this invention, the quantity of the formulation delivered, and the duration of administration of a single dose depend on the type of inhalation device employed. For some aerosol delivery systems, such as nebulizers, the frequency of administration and length of time for which the system is activated will depend mainly on the concentration of insulin conjugate in the aerosol. For example, shorter periods of administration can be used at higher concentrations of insulin conjugate in the nebulizer solution. Devices such as metered dose inhalers can produce higher aerosol concentrations, and can be operated for shorter periods to deliver the desired amount of insulin conjugate. Devices such as powder inhalers deliver active agent until a given charge of agent is expelled from the device. In this type of inhaler, the amount of insulin derivative of this invention in a given quantity of the powder determines the dose delivered in a single administration.

The particle size of insulin derivative of this invention in the formulation delivered by the inhalation device is critical with respect to the ability of insulin to make it into the lungs, and into the lower airways or alveoli. The insulin derivative of this invention can be formulated so that at least about 10% of the insulin conjugate delivered is deposited in the lung, for example about 10 to about 20%, or more. It is known that the maximum efficiency of pulmonary deposition for mouth breathing humans is obtained with particle sizes of about 2 μm to about 3 μm. When particle sizes are above about 5 μm pulmonary deposition decreases substantially. Particle sizes below about 1 μm cause pulmonary deposition to decrease, and it becomes difficult to deliver particles with sufficient mass to be therapeutically effective. Thus, particles of the insulin derivative delivered by inhalation have a particle size less than about 10 μm, for example in the range of about 1 μm to about 5 μm. The formulation of the insulin derivative is selected to yield the desired particle size in the chosen inhalation device.

Advantageously for administration as a dry powder, an insulin derivative of this invention is prepared in a particulate form with a particle size of less than about 10 μm, for example about 1 to about 5 μm. The particle size is effective for delivery to the alveoli of the patient's lung. The dry powder is largely composed of particles produced so that a majority of the particles have a size in the desired range. Advantageously, at least about 50% of the dry powder is made of particles having a diameter less than about 10 μm. Such formulations can be achieved by spray drying, milling, or critical point condensation of a solution containing insulin conjugate and other desired ingredients. Other methods also suitable for generating particles useful in the current invention are known in the art.

The particles are usually separated from a dry powder formulation in a container and then transported into the lung of a patient via a carrier air stream. Typically, in current dry powder inhalers, the force for breaking up the solid is provided solely by the patient's inhalation. In another type of inhaler, air flow generated by the patient's inhalation activates an impeller motor which deagglomerates the particles.

Formulations of insulin derivatives of this invention for administration from a dry powder inhaler typically include a finely divided dry powder containing the derivative, but the powder can also include a bulking agent, carrier, excipient, another additive, or the like. Additives can be included in a dry powder formulation of insulin conjugate, for example, to dilute the powder as required for delivery from the particular powder inhaler, to facilitate processing of the formulation, to provide advantageous powder properties to the formulation, to facilitate dispersion of the powder from the inhalation device, to stabilize the formulation (for example, antioxidants or buffers), to provide taste to the formulation, or the like. Advantageously, the additive does not adversely affect the patient's airways. The insulin derivative can be mixed with an additive at a molecular level or the solid formulation can include particles of the insulin conjugate mixed with or coated on particles of the additive. Typical additives include mono-, di-, and polysaccharides; sugar alcohols and other polyols, such as, for example, lactose, glucose, raffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitol, starch, or combinations thereof; surfactants, such as sorbitols, diphosphatidyl choline, or lecithin; or the like. Typically an additive, such as a bulking agent, is present in an amount effective for a purpose described above, often at about 50% to about 90% by weight of the formulation. Additional agents known in the art for formulation of a protein such as insulin analogue protein can also be included in the formulation.

A spray including the insulin derivatives of this invention can be produced by forcing a suspension or solution of insulin conjugate through a nozzle under pressure. The nozzle size and configuration, the applied pressure, and the liquid feed rate can be chosen to achieve the desired output and particle size. An electrospray can be produced, for example, by an electric field in connection with a capillary or nozzle feed. Advantageously, particles of insulin conjugate delivered by a sprayer have a particle size less than about 10 μm, for example in the range of about 1 μm to about 5 μm.

Formulations of insulin derivatives of this invention suitable for use with a sprayer will typically include the insulin derivative in an aqueous solution at a concentration of about 1 mg to about 20 mg of insulin conjugate per ml of solution. The formulation can include agents such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant, and, for example zinc. The formulation can also include an excipient or agent for stabilization of the insulin derivative, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulk proteins useful in formulating insulin conjugates include albumin, protamine, or the like. Typical carbohydrates useful in formulating insulin conjugates include sucrose, mannitol, lactose, trehalose, glucose, or the like. The insulin derivative formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of the insulin conjugate caused by atomization of the solution in forming an aerosol. Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between about 0.001 and about 4% by weight of the formulation.

Pharmaceutical compositions containing an insulin derivative according to the present invention may also be administered parenterally to patients in need of such a treatment. Parenteral administration may be performed by subcutaneous, intramuscular or intravenous injection by means of a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can be performed by means of an infusion pump. Further options are to administer the insulin nasally or pulmonally, for example in compositions, powders or liquids, specifically designed for the purpose.

Injectable compositions of the insulin derivatives of the invention can be prepared using the conventional techniques of the pharmaceutical industry which involve dissolving and mixing the ingredients as appropriate to give the desired end product. Thus, according to one procedure, an insulin derivative according to the invention is dissolved in an amount of water which is somewhat less than the final volume of the composition to be prepared. An isotonic agent, a preservative and a buffer is added as required and the pH value of the solution is adjusted—if necessary—using an acid, e.g. hydrochloric acid, or a base, e.g. aqueous sodium hydroxide as needed. Finally, the volume of the solution is adjusted with water to give the desired concentration of the ingredients.

In a further aspect of the invention the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof. Each one of these specific buffers constitutes an alternative aspect of the invention.

In a further aspect of the invention the formulation further comprises a pharmaceutically acceptable preservative which may be selected from the group consisting of phenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethonium chloride, chlorphenesine (3p-chlorphenoxypropane-1,2-diol) or mixtures thereof. In a further aspect of the invention the preservative is present in a concentration from 0.1 mg/ml to 20 mg/ml. In a further aspect of the invention the preservative is present in a concentration from 0.1 mg/ml to 5 mg/ml. In a further aspect of the invention the preservative is present in a concentration from 5 mg/ml to 10 mg/ml. In a further aspect of the invention the preservative is present in a concentration from 10 mg/ml to 20 mg/ml. Each one of these specific preservatives constitutes an alternative aspect of the invention. The use of a preservative in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19^(th) edition, 1995.

In a further aspect of the invention the formulation further comprises an isotonic agent which may be selected from the group consisting of a salt (e.g. sodium chloride), a sugar or sugar alcohol, an amino acid (e.g. glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), an alditol (e.g. glycerol (glycerine), 1,2-propanediol (propyleneglycol), 1,3-propanediol, 1,3-butanediol) polyethyleneglycol (e.g. PEG400), or mixtures thereof. Any sugar such as mono-, di-, or polysaccharides, or water-soluble glucans, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na may be used. In one aspect the sugar additive is sucrose. Sugar alcohol is defined as a C4-C8 hydrocarbon having at least one—OH group and includes, for example, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. In one aspect the sugar alcohol additive is mannitol. The sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to the amount used, as long as the sugar or sugar alcohol is soluble in the liquid preparation and does not adversely effect the stabilizing effects achieved using the methods of the invention. In one aspect, the sugar or sugar alcohol concentration is between about 1 mg/ml and about 150 mg/ml. In a further aspect of the invention the isotonic agent is present in a concentration from 1 mg/ml to 50 mg/ml. In a further aspect of the invention the isotonic agent is present in a concentration from 1 mg/ml to 7 mg/ml. In a further aspect of the invention the isotonic agent is present in a concentration from 8 mg/ml to 24 mg/ml. In a further aspect of the invention the isotonic agent is present in a concentration from 25 mg/ml to 50 mg/ml. Each one of these specific isotonic agents constitutes an alternative aspect of the invention. The use of an isotonic agent in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19^(th) edition, 1995.

Typical isotonic agents are sodium chloride, mannitol, dimethyl sulfone and glycerol and typical preservatives are phenol, m-cresol, methyl p-hydroxybenzoate and benzyl alcohol.

Examples of suitable buffers are sodium acetate, glycylglycine, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and sodium phosphate.

A composition for nasal administration of an insulin derivative according to the present invention may, for example, be prepared as described in European Patent No. 272097 (to Novo Nordisk A/S).

Compositions containing insulin derivatives of this invention can be used in the treatment of states which are sensitive to insulin. Thus, they can be used in the treatment of type 1 diabetes, type 2 diabetes and hyperglycaemia for example as sometimes seen in seriously injured persons and persons who have undergone major surgery. The optimal dose level for any patient will depend on a variety of factors including the efficacy of the specific insulin derivative employed, the age, body weight, physical activity, and diet of the patient, on a possible combination with other drugs, and on the severity of the state to be treated. It is recommended that the daily dosage of the insulin derivative of this invention be determined for each individual patient by those skilled in the art in a similar way as for known insulin compositions.

Where expedient, the insulin derivatives of this invention may be used in mixture with other types of insulin, e.g. insulin analogues with a more rapid onset of action. Examples of such insulin analogues are described e.g. in the European patent applications having the publication Nos. EP 214826 (Novo Nordisk A/S), EP 375437 (Novo Nordisk A/S) and EP 383472 (Eli Lilly & Co.).

The present invention is further illustrated by the following examples which, however, are not to be construed as limiting the scope of protection.

DESCRIPTION OF DRAWINGS

FIG. 1: Size-exclusion chromatography of the insulin derivative in example 2 in mixture with insulin aspart (B28Asp human insulin). Insulin content in each individual peak is quantified by HLPC. The insulin derivative and Aspart in formulations with either 2.1 Zn(II) per hexamer or 6 Zn(II) per hexamer elutes as two separate fractions (high-molecular weight insulin and medium molecular weight insulin, respectively). The SEC experiments are performed in accordance with example 20.

FIG. 2: Clamp action profile after subcutaneous injection of the insulin derivative described in example 2 in different concentrations and with different Zn(II) concentrations demonstrating that the action profile of the insulin derivative is similar whether the insulin derivative is administered with 2.3 or 6 Zn(II) per six insulin or as 600 μM or 1200 μM formulations. The clamp experiment is performed in accordance with example 21.

FIG. 3: Clamp action profile after subcutaneous injection of the insulin derivative described in example 2 in mixture with insulin Aspart, or as individual injections, demonstrating that there is no significant blunting of the individual insulin action profiles. The clamp experiment is performed in accordance with example 21.

FIG. 4: Clamp action profile after subcutaneous injection of the insulin derivative in example 2 in three doses demonstrating long effect of insulin action. The clamp experiment is performed in accordance with example 21.

EXAMPLES Example 1 Synthesis of N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-glutamylamide desB30 human insulin

Step 1: Mono-tert-butyl hexadecandioate

Hexadecadioic acid (40.0 g, 140 mmol) was suspended in toluene (250 ml) and the mixture was heated to reflux. N,N-dimethylformamide di-tert-butyl acetal (76.3 g, 375 mmol) was added drop-wise over 4 hours. The mixture was refluxed overnight. The solvent was removed in vacuo at 50° C., and the crude material was suspended in DCM/AcOEt (500 ml, 1:1) and stirred for 15 mins. The solids were collected by filtration and triturated with DCM (200 ml). The filtrated were evaporated in vacuo to give crude mono-tert-butyl hexadecandioate, 30 grams. This material was suspended in DCM (50 ml), cooled with ice for 10 mins, and filtered. The solvent was removed in vacuo to leave 25 gram crude mono-tert-butyl hexadecandioate, which was recrystallized from heptane (200 ml) to give mono-tert-butyl hexadecandioate, 15.9 g (33%).

¹H-NMR (CDCl₃) δ: 2.35 (t, 2H), 2.20 (t, 2H), 1.65-1.55 (m, 4H), 1.44 (s, 9H), 1.34-1.20 (m, 20H).

Step 2: Succinimidyl tert-butyl hexadecandioate

The mono tert-butyl ester (2 g, 5.8 mmol) was dissolved in THF (20 ml) and treated with TSTU (2.1 g, 7.0 mmol) and DIEA (1.2 ml, 7.0 mmol) and stirred overnight. The mixture was filtered, and the filtrate was evaporated in vacuo. The residue was dissolved in AcOEt and washed twice with cold 0.1 M HCl and water. Drying over MgSO₄ and evaporation in vacuo gave succinimidyl tert-butyl hexadecandioate, 2.02 g (79%).

¹H-NMR (CDCl₃) δ: 2.84 (s, 4H), 2.60 (t, 2H), 2.20 (t, 2H), 1.74 (p, 2H), 1.56 (m, 2H), 1.44 (s, 9H), 1.40 (m, 2H), 1.30-1.20 (m, 18H).

Step 3: ω-tert-butyl-carboxy-pentadecanoyl-L-glutamylamide

Succinimidyl tert-butyl hexadecandioate (100 mg, 0.227 mmol) was dissolved in DMF (2 ml) and treated with L-glutamylamide (37 mg, 0.25 mmol) and DIEA (58 μl, 0.34 mmol) and the mixture was stirred overnight. The solvent was evaporated in vacuo, and the crude product was dissolved in AcOEt, and washed twice with 0.2M HCl, with water and brine. Drying over MgSO₄ and evaporation in vacuo gave ω-tert-butyl-carboxy-pentadecanoyl-L-glutamyl amide, 85 mg (80%).

¹H-NMR (CDCl₃) δ: 6.98 (s, 1H), 6.60 (d, 1H), 5.88 (s, 1H), 4.69 (m, 1H), 2.55-2.41 (m, 2H), 2.25-2.18 (m, 2H), 2.14 (m, 1H), 1.93 (m, 1H), 1.65-1.54 (m, 4H) 1.44 (s, 9H), 1.27 (br, 20H).

Step 4: ω-tert-butyl-carboxy-pentadecanoyl-L-glutamylamide γ-succinimidyl ester

ω-tert-butyl-carboxy-pentadecanoyl-L-glutamylamide (85 g, 0.181 mmol) was dissolved in THF (1 ml) and treated with TSTU (65 g, 0.217 mmol) and DIEA (37 μl, 0.217 mmol) and stirred overnight. The mixture was filtered, and the filtrate was evaporated in vacuo. The residue was dissolved in AcOEt and washed twice with cold 0.1 M HCl and water. Drying over MgSO₄ and evaporation in vacuo gave ω-tert-butyl-carboxy-pentadecanoyl-L-glutamyl amide γ-succinimidyl ester, 91 mg (89%).

¹H-NMR (CDCl₃) δ: 6.59 (s, 1H), 6.41 (d, 1H), 5.56 (s, 1H), 4.62 (m, 1H), 3.02-2.94 (dd, 2H), 2.84 (s, 4H), 2.71-2.58 (m, 2H), 1.76 (m, 1H), 1.53-1.63 (m, 4H), 1.44 (s, 9H), 1.25 (br, 20H).

Step 5: N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-glutamylamide desB30 human insulin

DesB30 human insulin (500 mg, 0.088 mmol) was dissolved in 100 mM Na₂CO₃ (5 ml, pH 10.2) at room temperature. ω-Tert-butyl-carboxy-pentadecanoyl-L-glutamyl amide γ-succinimidyl ester (57 mg, 0.105 mmol), was dissolved in acetonitrile (5 ml) and subsequently added to the insulin solution. After 30 mins, 0.2 M methylamine (0.5 ml) was added. pH was adjusted by HCl to 5.5, and the isoelectric precipitate was collected by centrifugation and dried in vacuo to give 423 mg. The coupling yield was 42% (RP-HPLC, C4 column; Buffer A: 10% MeCN in 0.1% TFA-water, Buffer B: 80% MeCN in 0.1% TFA-water; gradient 20% to 90% B in 16 minutes). The protected product was dissolved in 95% TFA (12 ml), left 30 mins, and evaporated in vacuo. The crude product was dissolved in water and lyophilized.

N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-glutamylamide desB30 human insulin was purified by RP-HPLC on C4-column, buffer A: 20% EtOH+0.1% TFA, buffer B: 80% EtOH+0.1% TFA; gradient 15-60% B, followed by HPLC on C4-column, buffer A: 10 mM Tris+15 mM ammonium sulphate in 20% EtOH, pH 7.3, buffer B: 80% EtOH, gradient 15-60% B. The collected fractions were desalted on Sep-Pak with 70% acetonitrile+0.1% TFA, neutralized by addition of ammonia and freeze-dried. The unoptimized yield was 50 mg, 12%. The purity as evaluated by HPLC was >98%. LCMS 6102.8; C₂₇₄H₄₁₂N₆₆O₈₀S₆ requires 6103.1.

Example 2 Synthesis of N^(εB29)-ω-carboxy-pentadecanoyl-γ-amino-butanoyl desB30 human insulin

This compound was prepared from hexadecandioic acid and γ-aminobutyric acid, in analogy with example 1.

ω-tert-butyl-carboxy-pentadecanoyl-γ-amino-butyric acid succinimidyl ester

¹H-NMR (CDCl₃) δ: 5.80 (m, 1H), 3.36 (dd, 2H), 2.84 (s, 4H), 2.65 (t, 2H), 2.21-2.13 (m, 4H), 1.99 (p, 2H), 1.44 (s, 9H), 1.66-1.51 (m, 6H), 1.25 (br, 20H).

N^(εB29)-ω-carboxy-pentadecanoyl-γ-amino-butanoyl desB30 human insulin

LCMS 6059.9; C₂₇₃H₄₁₁N₆₅O₇₉S₆ requires 6060.1.

Example 3 Synthesis of N^(εB29)-ω-carboxy-tetradecanoyl-γ-L-glutamylamide desB30 human insulin

This compound was prepared from pentadecandioic acid and L-glutamylamide in analogy with example 1.

LCMS 6088.2; C₂₇₃H₄₁₀N₆₆O₈₀S₆ requires 6089.1.

Example 4 Synthesis of N^(εB29)-ω-carboxy-tridecanoyl-γ-L-glutamylamide desB30 human insulin

This compound was prepared from tetradecandioic acid and L-glutamylamide in analogy with example 1.

LCMS 6075.3; C₂₇₂H₄₀₈N₆₆O₈₀S₆ requires 6075.1.

Example 5 Synthesis of N^(εB29)-ω-carboxy-pentadecanoyl-β-alanyl desB30 human insulin

This compound was prepared from hexadecandioic acid and β-alanine in analogy with example 1.

LCMS 6044.8; C₂₇₂H₄₀₉N₆₅O₇₉S₆ requires 6046.1.

Example 6 Synthesis of N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-aspartylamide desB30 human insulin

This compound was prepared from hexadecandioic acid and L-aspartylamide in analogy with example 1.

LCMS 6088.8; C₂₇₃H₄₁₀N₆₆O₈₀S₆ requires 6089.1.

Example 7 Synthesis of N^(εB29)-ω-carboxy-pentadecanoyl-ε-aminohexanoyl desB30 human insulin

This compound was prepared from hexadecandioic acid and ε-amino-hexanoic acid in analogy with example 1.

LCMS 6086.1; C₂₇₅H₄₁₅N₆₅O₇₉S₆ requires 6088.1.

Example 8 Synthesis of N^(εB29)-ω-carboxy-pentadecanoyl-δ-aminopentanoyl desB30 human insulin

This compound was prepared from hexadecandioic acid and δ-amino-pentanoic acid in analogy with example 1.

LCMS 6074.2, C₂₇₄H₄₁₃N₆₅O₇₉S₆ requires 6074.1.

Example 9 Synthesis of N^(εB29)-10-(4-carboxyphenoxy)-decanoyl-γ-L-glutamylamide desB30 human insulin

Step 1: 4-Hydroxy-benzoic acid tert-butyl ester

4-Hydroxy-benzoic acid (3 g, 21.7 mmol) was stirred in toluene (35 ml, dried over mol. sieves). The solution was heated to 80° C. under N₂, and N,N′-dimethylformamide di-tert-butyl acetal (10.42 mL, 43.4 mmol) was added over ca. 5 min. The mixture was stirred at 80° C. for 1 h 10 min., and cooled to rt. The solution was washed with water, twice with sat. NaHCO₃ and sat. NaCl (15 mL each), dried over MgSO₄, and concentrated to yield a yellow oil (2.77 g). The product was purified by flash chromatography (380 g silica, eluant: 4:6 AcOEt/heptane (2 L) and 1:1 AcOEt/heptane 700 mL) to yield white crystals (2.07 g, 49% yield).

HPLC-MS m/z: 217 (M+23).

¹H-NMR (CDCl₃, 400 MHz) 7.90 (d, 2H), 6.85 (d, 2H), 6.10 (s, 1H), 1.59 (s, 9H).

Step 2: 4-(9-Methoxycarbonylnonyloxy)benzoic acid tert-butyl ester

4-Hydroxy-benzoic acid tert-butyl ester (500 mg, 2.57 mmol) and 10-bromodecanoic acid methyl ester (683 mg, 2.57 mmol) were dissolved in acetonitrile, and K₂CO₃ (712 mg, 5.15 mmol) was added. The mixture was refluxed under nitrogen for 16 h, and cooled to rt. The solids were filtered off, and the filtrate concentrated under vacuum. The resulting residue was dissolved in AcOEt (50 ml) and water (25 mL). The phases were separated and the organic phase was dried over MgSO₄ and concentrated to yield a colorless oil (874 mg, 90% yield).

HPLC-MS m/z: 402 (M+23).

¹H-NMR (CDCl₃, 400 MHz) 7.92 (d, 2H), 6.87 (d, 2H), 3.99 (t, 2H), 3.67 (s, 3H), 2.31 (t, 2H), 1.72-1.83 (m, 2H), 1.59-1.69 (m, 2H), 1.58 (s, 9H), 1.40-1.50 (m, 2H), 1.23-1.40 (br, 8H).

Step 3: 4-(9-Carboxynonyloxy)benzoic acid tert-butyl ester

4-(9-Methoxycarbonylnonyloxy)benzoic acid tert-butyl ester (858 mg, 2.27 mmol) was dissolved in THF (5 ml), and 1N NaOH (2.27 mmol) was added. The mixture was stirred for 16 h. AcOEt (40 mL) and 1N HCl (2.38 ml) in water (25 ml) were added. The phases were separated, and the organic phase was dried over MgSO₄, and concentrated under vacuum to yield a white solid (781 mg, 95% yield).

HPLC-MS m/z: 387 (M+23).

¹H-NMR (CDCl₃, 400 MHz) 7.92 (d, 2H), 6.87 (d, 2H), 3.99 (t, 2H), 2.35 (t, 2H), 1.73-1.84 (m, 2H), 1.60-1.69 (m, 2H), 1.58 (s, 9H), 1.39-1.51 (m, 2H), 1.24-1.39 (br, 8H).

Step 4: 4-[9-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)nonyloxy]benzoic acid tert-butyl ester

4-(9-Carboxynonyloxy)benzoic acid tert-butyl ester (779 mg, 2.14 mmol) was dissolved in THF (15 mL), and DIEA (366 μl, 2.14 mmol) was added. The mixture was cooled to 0° C., and placed under nitrogen, and HSTU was added. The mixture was stirred at 0° C. for 30 min and at RT for 16 h. The sample was concentrated under vacuum and AcOEt (40 ml) was added. The mixture was washed with 0.2 N HCl (2×25 ml), dried over MgSO₄, and concentrated under vacuum to yield a slightly yellow solid. The solid was recrystallized from AcOEt to yield a white powder (276 mg, 28% yield). The mother liquor was concentrated to yield crystalline residue (430 mg, 43% yield). Data for the white powder:

HPLC-MS m/z: 484 (M+23).

¹H-NMR (CDCl₃, 300 MHz) 7.93 (d, 2H), 6.88 (d, 2H), 3.99 (t, 2H), 2.83 (s, 4H), 2.61 (t, 2H), 1.67-1.88 (m, 4H), 1.58 (s, 11H, theoret. 9H+ water), 1.27-1.52 (m, 10H).

Step 5: 4-[9-((S)-1-Carbamoyl-3-carboxypropylcarbamoyl)nonyloxy]benzoic acid tert-butyl ester

4-[9-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)nonyloxy]benzoic acid tert-butyl ester (200 mg, 0.433 mmol) was stirred in DMF (2 mL) and H-Glu-NH₂ (63 mg) was added. The non-homogeneous mixture was stirred at rt for 16 h. LC/MS analysis indicated the reaction had not gone to completion. H-Glu-NH₂ (20 mg) and more DMF (2 mL) were added and the mixture was stirred for 2 d at rt. The sample was concentrated under vacuum and AcOEt (50 mL) was added. The solution was washed with 0.2 N HCl (2×25 mL) and water (25 mL), dried over MgSO₄, and concentrated under vacuum to yield a white solid (180 mg, 86% yield).

HPLC-MS m/z: 493 (M+1).

Step 6: 4-{9-[(S)-1-Carbamoyl-3-(2,5-dioxopyrrolidin-1-yloxycarbonyl)propylcarbamoyl]nonyloxy}benzoic acid tert-butyl ester

The HSTU activation was performed in manner similar to that described for 4-[9-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)nonyloxy]benzoic acid tert-butyl ester. The product was purified by flash chromatography (1:1 AcOEt:heptane and AcOEt) to yield 18 mg.

HPLC-MS m/z: 590 (M+1).

Step 7: N^(εB29)-10-(4-carboxyphenoxy)decanoyl-γ-L-glutamylamide desB30 insulin

DesB30 insulin (126 mg, 0.022 mmol) was dissolved by adding 100 mM Na₂CO₃ (1.5 mL) and acetonitrile (1.5 mL) in a 10 ml round bottom-flask. 4-{9-[(S)-1-Carbamoyl-3-(2,5-dioxopyrrolidin-1-yloxycarbonyl)propylcarbamoyl]nonyloxy}benzoic acid tert-butyl ester (14 mg, 0.022 mmol) was added in acetonitrile (750 uL) and Na₂CO₃ (750 uL) was added so the final solution was 50:50 100 mM Na₂CO₃/acetonitrile. The solution was stirred at RT for 1 h. The solution was transferred to a 15 ml centrifuge tube, washing with Milli-Q water (6 ml). The solution was cooled on ice, and the pH was adjusted to 5.1 by adding 1N HCl, which lead to precipitation. The tube was centrifuged at 5000 rpm for 10 min at 10° C. The solvent was decanted from the solid. 95:5 TFA/water (2.5 ml) was added to the solid. The solution was poured into a round bottom flask, washing with more 95:5 TFA/water (2.5 ml). The solution was stirred for 30 min at RT, and concentrated under vacuum. DCM was added and removed twice, and the flask was placed under vacuum at RT. The product was purified by preparative HPLC (C18 column, acetonitrile/water/0.05% TFA). The relevant fractions were pooled (two batches) and diluted 1:1 with water. The solutions were cooled on ice, and the precipitation was induced by adjusting the pH to ca. 5 with 1 N NaOH. The samples were centrifuged (5000 rpm, 10 min, 5° C.). The liquid was decanted off and the pellets were lyophilized to yield a white solid (22 mg+12 mg).

MALDI-MS (alpha-cyano-4-hydroxycinnamic acid) m/z: 6128.7 (M=6125.1).

HPLC-MS m/z: 1532.8 ((M+4)/4=1532.2).

Example 10 Synthesis of N^(εB29)-4-[11-(4-Carboxyphenyl) undecanoylamino]butyryl desB30 human insulin

Step 1: 4-Iodobenzoic acid tert-butyl ester

4-Iodobenzoic acid (10 g, 40.3 mmol) was dissolved in dry toluene (100 ml, dried over mol. sieves). The solution was heated to 70° C. under a flow of nitrogen. A solution of N,N′-dimethylformamide di-tert-butyl acetal (24.6 g, 121 mmol) in toluene (25 mL) was added over ca. 30 min. The reaction was mixed for 16 h. At some point the heating unit failed, so the reaction cooled from 70° C. to rt. The solution was heated to 70° C. for and mixed for 5 h. The sample was concentrated under vacuum, and AcOEt (400 ml) was added. The solution was then washed with 1:1 sat. NaHCO₃/water (150 ml), and sat. NaHCO₃, water and sat. NaCl (75 mL each). The organic phase was dried (MgSO₄) and concentrated under vacuum to yield light brown oil.

HPLC-MS m/z: 327 (M+23).

¹H-NMR (CDCl₃, 400 MHz) δ 7.77 (d, 2H), 7.69 (d, 2H), 1.58 (s, 9H).

Step 2: 11-Iodo undecanoic acid methyl ester

11-Bromo undecanoic acid methyl ester (20.2 g, 72.3 mmol) was dissolved in acetone (200 ml). Sodium iodide (54 g, 361 mmol) was added and reaction was refluxed under nitrogen for 16 h. After cooling to RT the salts were filtered off. The filtrate was concentrated under vacuum and water (200 ml) was added. The solution was extracted with AcOEt (2×100 ml) adding some sat. NaCl to aid phase separation. The organic extracts were pooled and washed with water (100 ml) plus a little sat. NaCl, and sat. NaCl (50 mL). Dry over MgSO₄. The solution was a red-orange color. Three teaspoons of activated charcoal were added. After mixing, the solution was filtered through a bed of celite. The filtrate was concentrated under vacuum to yield a light yellow oil (20.96 g, 89%).

HPLC-MS m/z: 327 (M+1).

¹H-NMR (CDCl₃, 300 MHz) δ 3.67 (s, 3H), 3.19 (t, 2H), 2.30 (t, 2H), 1.74-1.89 (m, 2H), 1.53-1.70 (m, 2H), 1.34-1.46 (m, 2H), 1.28 (br, 10H).

Step 3: 4-(10-Methoxycarbonyldecyl)benzoic acid tert-butyl ester

All glassware was dried prior to use. THF was dried over molecular sieves. LiCl was dried at 150° C. for 1 h, then stored in a closed bottle. All reaction solutions were made under nitrogen, and the solutions were transferred via syringe. 4-Iodobenzoic acid tert-butyl ester (1.2 g, 3.95 mmol) was dissolved in THF (3 ml) and cooled to −30° C. Isopropyl magnesium chloride (4.34 mmol, 2M in THF) was added over 5 minutes, and the solution was stirred for 1 hr at a temperature between −18° C. to −25° C. The solution was cooled to −22° C., and a mixture of CuCN (0.389 g, 4.34 mmol) and LiCl (0.368 g, 8.68 mmol) in THF (4.2 ml) was then added. The reaction vessel was removed from cooling and allowed to warm to RT (ca. 10 min). Trimethylphosphite (0.95 mL) was added, and after stirring for 5 min at rt, a solution of 11-iodo-undecanoic acid methyl ester (1.0 g, 3.16 mmol) in THF (3 ml) was added. The solution was mixed at rt for 16 h. Sat. NH₄Cl (3 ml) was added, and the solution was poured into water (60 mL). The solution was extracted with AcOEt (3×35 ml). The organic extracts were pooled and washed with water (30 mL) using some Sat. NaCl to aid phase separation. The solvent was removed under vacuum to yield a biphasic residue. AcOEt (ca 2 ml) was added and the flask was swirled gently. Not all of the thick white residue dissolved. The portion which dissolved was added to a column of silica (50 g) and eluted with AcOEt: heptane 1:11. The appropriate fractions were concentration under vacuum to yield an oil (1.25 g). The oil was dissolved in acetone (30 mL), and piperidine (1 mL) was added. NaI (0.8 g) was added and the mixture was stirred and refluxed for 16 h. The mixture was concentrated under vacuum and partitioned between AcOEt (50 mL) and 1 N HCl (25 mL). The organic phase was washed with 1 N HCl (2×25 mL), dried over MgSO₄, and concentrated under vacuum to yield a colorless oil (1.1 g). The product was purified by flash chromatography (eluant: AcOEt:heptane 1:11, 150 g silica) to yield a colorless oil (0.72 g, 61%).

HPLC-MS m/z: 399 (M+23).

¹H-NMR (CDCl₃, 300 MHz) δ 7.90 (d, 2H), 7.21 (d, 2H), 3.66 (s, 3H), 2.64 (t, 2H), 2.30 (t, 2H), 1.48-1.70 (m, 13H), 1.27 (br, 12H).

Step 4: 4-(10-Carboxydecyl)benzoic acid tert-butyl ester

The compound was prepared in analogous fashion to the procedure used in the preparation of 4-(9-Carboxynonyloxy)benzoic acid tert-butyl ester to yield a white solid (0.68 g).

HPLC-MS m/z: 385 (M+23).

¹H-NMR (CDCl₃, 300 MHz) δ 7.90 (d, 2H), 7.21 (d, 2H), 2.64 (t, 2H), 2.34 (t, 2H), 1.53-1.71 (m, 13H), 1.28 (br, 12H).

Step 5: 4-[10-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)decyl]benzoic acid tert-butyl ester

The compound was prepared in analogous fashion to the procedure used in the preparation of 4-[9-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)nonyloxy]benzoic acid tert-butyl ester.

HPLC-MS m/z: 482 (M+23).

¹H-NMR (CDCl₃, 400 MHz) δ 7.89 (d, 2H), 7.21 (d, 2H), 2.76-2.93 (m, 4H), 2.54-2.68 (m, 2H), 1.67-1.81 (M, 2H), 1.52-1.66 (m, 11H), 1.35-1.43 (M, 2H), 1.19-1.35 (br, 10H).

Step 6: 4-[10-(3-Carboxy-propylcarbamoyl)decyl]benzoic acid tert-butyl ester

4-[10-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)decyl]benzoic acid tert-butyl ester (300 mg, 0.65 mmol) was dissolved in DMF (3 ml) and 4-amino butyric acid (67 mg, 0.65 mmol). The mixture was stirred for 16 h under nitrogen. The solvent was removed under vacuum and AcOEt (35 ml) was added. The solution was washed with 0.2 N HCl and water (15 ml each). Sat. NaHCO₃ was added (not intended) to the organic phase. DCM (50 ml) was added. Some of the organic phase was removed and DCM (100 ml) was added to the aqueous phase and allowed to stand overnight. The mixture was cooled on ice and the pH was adjusted to 1.9 with 4N HCl. The organic phase was isolated, dried over MgSO₄ and concentrated under vacuum to yield on oil (220 mg, 76% yield).

HPLC-MS m/z: 470 (M+23).

¹H-NMR (CDCl₃, 400 MHz) δ 7.89 (d, 2H), 7.21 (d, 2H), 5.79 (br, 1H), 3.27-3.40 (m, 2H), 2.64 (t, 2H), 2.40 (t, 2H), 2.18 (t, 2H), 1.78-1.91 (m, 2H), 1.51-1.61 (m, 13H), 1.35-1.43 (M, 2H), 1.17-1.36 (br, 12H).

Step 7: 4-{10-[3-(2,5-Dioxo-pyrrolidin-1-yloxycarbonyl)propylcarbamoyl]decyl}benzoic acid tert-butyl ester

The compound was prepared in analogous fashion to the procedure used in the preparation of 4-[9-(2,5-Dioxopyrrolidin-1-yloxycarbonyl)nonyloxy]benzoic acid tert-butyl ester, but TSTU was used instead of HSTU. Precipitation (DCM/Heptane) yielded white crystals (180 mg, 70% yield).

HPLC-MS m/z: 568 (M+23).

¹H-NMR (CDCl₃, 400 MHz) δ 7.89 (d, 2H), 7.21 (d, 2H), 5.83 (br, 1H), 3.30-3.43 (m, 2H), 2.85 (br, 4H), 2.57-2.73 (m, 4H), 2.15 (t, 2H), 1.92-2.07 (m, 2H), 1.56-1.64 (m, 13H), 1.18-1.36 (br, 12H).

Step 8: N^(εB29)-4-[11-(4-carboxyphenyl)undecanoylamino]butyryl desB30 human insulin

The compound was prepared in analogous fashion to the procedure used in the preparation of example 9 to yield 30 mg.

MALDI-MS (alpha-cyano-4-hydroxycinnamic acid) m/z: 6067 (M=6080, reference standard (M=5706) showed M−13).

HPLC-MS m/z: 1520.9 ((M+4)/4=1521).

Example 11 Synthesis of N^(εB29)-(3-(3-{4-[3-(7-carboxyheptanoylamino)propoxy]butoxy}propylcarbamoyl)-propionyl-γ-glutamylamide) desB30 human Insulin

Step 1: N-{3-[4-(3-tert-Butoxycarbonylaminopropoxy)-butoxy]-propyl}succinamic acid

123-0000-3007

1-(tert-Butoxycarbonylamino)-4,9-dioxa-12-dodecanamine (5.0 g, 16.45 mmol) was dissolved in THF (30 mL), succinic anhydride (1.81 g, 18.1 mmol) in acetonitrile (10 mL) was added and the mixture was heated to 60 C for 4 h, and subsequently stirred at RT overnight.

The mixture was evaporated to dryness and EtAc (50 mL) was added.

The EtAc phase was washed with HCl (0.1 M) 3 times, dried with MgSO₄ and subsequently the organic phase was evaporated to dryness which gave 5.86 g (88%) of thick oil.

LCMS: Rt 2.86 min; m/z (M+1) 405. Calcd: 405.

This product was used without further purification.

Step 2: Octanedioic acid tert-butyl ester 2,5-dioxo-pyrrolidin-1-yl ester

Octanedioic acid mono-tert-butyl ester (3.14 g, 13.63 mmol) was dissolved in THF (100 mL). TSTU (4.9 g, 16.3 mmol) was added and pH was adjusted to 8.5 with DIPEA (2.85 mL).

The mixture was stirred under nitrogen overnight, evaporated to dryness, dissolved in EtAc (50 mL) which subsequently was extracted 2 times with HCL (0.1 M). The organic phase was dried with MgSO₄, filtered and evaporated resulting in an slightly yellow oil (5 g, containing small amounts of solvent).

LCMS: Rt 6.56 min; m/z (M+1) 328. Calcd: 328.

Step 3: 7-(3-{4-[3-(3-carboxypropionylamino)propoxy]butoxy}propylcarbamoyl)heptanoic acid tert-butyl ester

123-0000-3012

N-{3-[4-(3-tert-Butoxycarbonylaminopropoxy)-butoxy]-propyl}succinamic acid (4.60 g, 11.37 mmol) was stirred with TFA (20 mL) at RT for 60 min, after evaporation the residue was stripped with DCM (30 mL×2) and evaporated to dryness.

The resulting oil was dissolved in acetonitrile (30 mL) and octanedioic acid tert-butyl ester 2,5-dioxo-pyrrolidin-1-yl ester (4.46 g, 13.6 mmol) in DMF (20 mL) was added.

pH was adjusted to 8.5 with DIPEA and the mixture was stirred overnight under nitrogen. The mixture was subsequently evaporated to dryness and redissolved in EtOAc (50 mL). The EtOAc phase was extracted ×3 with HCl (0.1 M), the organic layer dried over magnesium sulphate, filtered and evaporated resulting in a slightly yellow crystalline oil (6.5 g, content of solvent residues).

LCMS: Rt 4.31 min; m/z (M+1) 517. Calcd: 517.

The crude product was used for further reaction without further purification.

Step 4: 7-[3-(4-{3-[3-((S)-1-carbamoyl-3-carboxypropylcarbamoyl)propionylamino]propoxy}butoxy)propylcarbamoyl]heptanoic acid tert-butyl ester

0123-0000-3078

7-(3-{4-[3-(3-Carboxypropionylamino)propoxy]butoxy}propylcarbamoyl)heptanoic acid tert-butyl ester (2.4 g), the crude product from step 3, was dissolved in THF (60 mL), TSTU (2.11 g, 6.97 mmol) was added together with DMF (10 mL), pH was adjusted to 8.2 with DIPEA (0.8 mL). The mixture was stirred overnight under nitrogen.

The mixture was evaporated and the residue dissolved in EtOAc which was extracted with HCl (0.1 M) 3 times. The organic layer was dried with magnesium sulphate, filtered and the filtrate evaporated to give 3.2 g oil.

LCMS: Rt 4.57 min; m/z 614, corresponding to the activated acid.

This crude product was dissolved in acetonitrile (40 mL) and L-glutamic acid amide (0.6 g, 4.1 mmol) was added together with DMF (5 mL), pH was adjusted to 8.2 with DIPEA (1.4 mL).

The mixture was stirred at RT for overnight; filtration followed by evaporation afforded thick yellow oil.

This was extracted between EtOAc and HCl (0.1 M) as reported above, and the resulting dried EtOAc layer gave 1.66 g crude product on evaporation.

LCMS: Rt 3.62 min; m/z (M+1) 645. The crude product was purified by preparative HPLC using acetonitrile/water/0.1% TFA as eluent on C18 column (Jones, Kromasil RP18 5 μm 15×225 mm). Gradient: 0.0-10.0 min 35% acetonitrile A; 10.0-30.0 min 35-90% A; The product was collected in fractions from 16-18 min. The combined fractions were evaporated yielding the wanted product (1.0 g). LCMS: Rt 3.59 min; m/z (M+1) 645, calcd. 645.

Step 5: N^(εB29)-(3-(3-{4-[3-(7 carboxyheptanoylamino)propoxy]butoxy}propylcarbamoyl)propionyl-γ-L-glutamylamide) desB30 human insulin

7-[3-(4-{3-[3-((S)-1-Carbamoyl-3-carboxypropylcarbamoyl)propionylamino]propoxy}butoxy)-propylcarbamoyl]heptanoic acid tert-butyl ester from step 4 (1.0 g, 1.55 mmol) was dissolved in THF (20 mL), TSTU (0.51 g, 1.7 mmol) was added and pH adjusted to >8 with DIPEA (0.27 mL).

The mixture was stirred overnight under nitrogen. Evaporation followed by extraction between EtOAc and HCl (0.1 M), drying of the organic phase (MgSO₄) followed by evaporation to dryness gave 21 mg oil, LCMS: Rt. 4.34 min, m/z 742.

This crude product was dissolved in acetonitrile (10 mL), pH was adjusted to 8 with Na₂CO₃ (0.1 M) and added to a solution of desB30 human insulin (1 g) dissolved in Na₂CO₃ solution (15 mL, pH 10.2).

The mixture was stirred under nitrogen at RT for 1 h. Then pH was adjusted to 5.4 by means of HCl (2M) resulting in precipitation. The mixture was filtered, the filtrate freeze dried, and the precipitate dried in vacuum overnight.

Both fractions were purified on Gilson using acetonitrile/water/0.1% TFA as eluent on C18 column (Jones, Kromasil RP18 5 μm 15×225 mm).

Gradient: 0.0-5.0 min 35% acetonitrile (A); 5.0-25.0 min 35-90% A; The product was collected in fractions from 12-15 min. The combined fractions were evaporated, redissolved in water and freeze-dried yielding 27 mg of the wanted product.

LCMS: Rt. 7.76 min, m/z 1570

MALDI-MS (sinnapinic acid): 6277; C₂₈₀H₄₂₂N₆₈O₈₄S₆ requires 6277.

Example 12 Synthesis of N^(εB29)-ω-carboxy-tridecanoyl-γ-amino-butanoyl desB30 human insulin

This compound was prepared from tetradecandioic acid and γ-amino-butyric acid in analogy with example 1.

LCMS 6032.1, C₂₇₁H₄₀₇N₆₅O₇₉S₆ requires 6032.0.

Example 13 Synthesis of N^(εB29)-ω-carboxy-undecanoyl-δ-amino-butanoyl desB30 human insulin

This compound was prepared from dodecandioic acid and γ-amino-butyric acid in analogy with example 1.

LCMS 6003.8, C₂₆₉H₄₀₃N₆₅O₇₉S₆ requires 6004.0.

Example 14 Synthesis of N^(εB29)-ω-carboxy-tetradecanoyl-γ-amino-butanoyl desB30 human insulin

This compound was prepared from pentadecandioic acid and γ-aminobutyric acid in analogy with example 1.

LCMS 6045.6, C₂₇₂H₄₀₉N₆₅O₇₉S₆ requires 6046.1.

Example 15 Synthesis of N^(εB29)-{4-[10-(4-Carboxy-phenoxy)-decanoylamino]-butyryl} desB30 insulin

This compound was prepared from 4-(9-methoxycarbonylnonyloxy)benzoic acid tert-butyl ester in analogy with examples 9 and 10.

LCMS: 6095.6, C₂₇₆H₄₀₉N₆₅O₇₉S₆ requires 6094.1.

Example 16 Synthesis of N^(εB29)-{4-[(14-Carboxy-tetradecanoylamino)-methyl]-benzoyl} desB30 insulin

This compound was prepared from 4-aminomethyl benzoic acid in analogy with example 1.

LCMS: 6082.0, C₂₇₅H₄₀₆N₆₆O₈₁S₆ requires 6082.1.

Example 17 Synthesis of N^(εB29)-[16-(4-Carboxy-phenoxy)-hexadecanoyl] desB30 insulin

Step 1: 16-Bromohexadecanoic acid methyl ester

16-Bromohexadecanoic acid (6 g, 17.9 mmol) was dissolved in methanol (35 mL), toluene (100 mL) and trimethylorthoformate (20 mL). Amberlyst 15 was added and the mixture was stirred under nitrogen for 16 h at 55° C. The mixture was concentrated, and dissolved in methanol (ca. 50 mL) and DCM (30 mL). The resin was filtered off, and the filtrate was concentrated. The volume was increased to ca. 40 mL with methanol. Cooling produced crystals which were filtered off, washed with cold methanol and dried to yield white crystals (5.61 g, 90% yield).

¹H-NMR (DMSO, 300 MHz) 3.57 (s, 3H), 3.52 (t, 2H), 2.28 (t, 2H), 1.78 (m, 2H), 1.50 (m, 2H), 1.37 (m, 2H).

The remainder of the steps were performed in analogy with example 9.

LCMS: 6081.2, C₂₇₆H₄₁₀N₆₄O₇₉S₆ requires 6081.1.

Example 18 Synthesis of N^(εB29)-{4-[(15-carboxypentadecanoylamino)benzoyl]-desB30 human insulin

Step 1: 4-(15-tert-Butoxycarbonylpentadecanoylamino)-benzoic acid

Mono-tert-butyl hexadecandioate hexadecadioic acid (400 mg, 1.17 mmol) was dissolved in NMP (6 ml). 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (223 mg, 1.17 mmol) and 1-hydroxy-7-azabenzotriazole (156 mg, 1.17 mmol) was added and the mixture was heated to 50 degrees celcius for 90 min. 4-aminobenzoic acid (320 mg, 2.34 mmol) and DIEA (0.6 mL, 3.51 mmol) was added and the mixture was stirred under nitrogen flow overnight. The mixture is transferred is separated between saturated aqueous NaHCO₃ (50 mL) and diethyl ether (100 mL). NaHSO₄ (50 mL, 10% in water) was added and the organic phase isolated, dried (MgSO₄) and solvent removed in vacuo. The crude product was recrystallized from ethanol to give 4-(15-tert-butoxycarbonylpentadecanoylamino)-benzoic acid.

¹H-NMR (DMSO-d₆) δ: 10.18 (s, 1H), 7.87 (d, 2H), 7.69 (d, 2H), 2.33 (t, 2H), 2.15 (t, 2H), 1.58 (t, 2H), 1.46 (t, 2H), 1.38 (s, 9H)_(m) 1.31-1.20 (m, 20H).

Step 2: 4-(15-tert-Butoxycarbonyl-pentadecanoylamino)-benzoic acid 2,5-dioxo-Pyrrolidin-1-yl ester

4-(15-tert-Butoxycarbonylpentadecanoylamino)-benzoic acid (29 mg, 0.063 mmol) was converted to the succinimidyl ester using TSTU similar to described above.

HPLC-MS, m/z=559 (M+23, M+Na).

Step 3: N^(εB29)-{4-[(15-carboxypentadecanoylamino)benzoyl]-desB30 human insulin

DesB30 human insulin (355 mg, 0.062 mmol) was dissolved in DMSO (3.5 mL), triethylamin (0.622 mmol, 0.087 mL) was added. 4-(15-tert-Butoxycarbonyl-pentadecanoylamino)-benzoic acid 2,5-dioxo-pyrrolidin-1-yl ester (28.3 mg, 0.051 mmol) was dissolved in DMSO (0.5 mL and added). The mixture was carefully stirred for 30 minutes at room temperature. The mixture was frozen by cooling with an icebath and water (7 mL) was added and the mixture left standing at room temperature until the frozen mixture had dissolved. pH was adjusted to pH=5.3 using 1 N HCl and precipitate isolated by centrifuge, washed once with water followed by centrifuge. Trifluoroacetic acid (15 mL) was added and the mixture was stirred for 30 minutes, poured into diethyl ether (50 mL) while maintaining cooling on an icebath. The crude product was isolated by centrifuge and dissolved in 10 mM TRIS+15 mM (NH₄)₂SO₄ in 20% EtOH, pH 7.3 and subjected to purification on an ÄKTA purifier employing a reversed phase HPLC, Jupiter 5269, C4 250/20 mm, 15 μM, 300 Å. The buffer consisted of A-buffer 10 mM TRIS+15 mM (NH₄)₂SO₄ in 20% EtOH, pH 7.3 and a B-buffer 80% EtOH. The product was eluted with a gradient 15-60% B with 8 ml/min. Fractions containing product were collected an pH adjusted to pH=5.2. N^(εB29)-{4-[(15-carboxypentadecanoylamino)benzoyl]-desB30 human insulin was isolated by centrifuges and lyophilized.

LCMS: 6092.0, C₂₇₆H₄₀₉N₆₅O₇₉S₆ requires 6094.1.

Example 19 Synthesis of N^(εB29)-{4-[(15-Carboxy-pentadecanoylamino)-methyl]-benzoyl}-desB30 insulin

Step 1: 4-[(15-tert-Butoxycarbonylpentadecanoylamino)methyl]benzoic acid

Hexadecanedioic acid tert-butyl ester 2,5-dioxo-pyrrolidin-1-yl ester (370 mg, 0.842 mmol) was dissolved in NMP (8 mL), 4 aminomethyl benzoic acid (127.2 mg, 0.842 mmol) was added, the mixture was stirred at room temperature overnight, followed by heating at for 2 hours at 50 degrees celcius. After cooling to room temperature the mixture was poured into water. Product isolated by filtration dried and used for the next step without any further purification.

¹H-NMR (DMSO-d₆) (selected signals) δ: 7.88 (d, 2H), 7.34 (d, 2H), 4.31 (d, 2H), 2.15 (m, 4H), 1.47 (m, 4H), 1.38 (s, 9H), 1.23 (br s, 2H), 1.38 (s, 9H).

HPLC-MS: m/z=498 (M+Na)

Step 2: N^(εB29)-{4-[(15-Carboxy-pentadecanoylamino)-methyl]-benzoyl}-desB30 insulin

The 4-[(15-tert-Butoxycarbonyl pentadecanoylamino)methyl]benzoic acid was converted to converted to the succinimidyl ester and reacted with DesB30 human insulin similar to described above. The product was purified on an ÄKTA purifier employing a reversed phase HPLC, Jupiter 5269, C4 250/20 mm, 15 μM, 300 Å. The buffer consisted of A-buffer 10 mM TRIS+15 mM (NH₄)₂SO₄ in 20% EtOH, pH 7.3 and a B-buffer 80% EtOH. The product was eluted with a gradient 15-60% B with 8 ml/min. Followed by purification with RP-HPLC on a Waters Prep LC2000, on C18, 5 cm×20 cm, flow 20 ml/min using acetonitrile/water 36-42% gradient containing 0.1% TFA. Fractions containing product was collected and lyophilized. To the lyophilized material was added water (7.2 mL) and pH adjusted to 8.98 with 1 N+0.1 N NaOH. The pH was adjusted back to 5.2-5.5 with 0.1 N HCl. The product precipitated, isolated by centrifuge and lyophilized to give the title compound.

LCMS: 6108.0, C₂₇₇H₄₁₁N₆₅O₇₉S₆ 6108.1

Example 20 Hydrophobicity Albumin Affinity Self-Association and Mixability of Long.Acting and Short-Acting Insulins

Analysis of Selv-Associating Properties of the Insulin Derivatives of the Invention

The ability of the insulin derivatives of the invention to self-associate into large, but soluble complexes is analysed using SEC (size exclusion chromatography):

Column: Superose ™ 6 PC 3.2/30, CV = 2.4 ml (Amerham Biosciences) Temperature: 37° C. SEC buffer: 140 mM NaCl, 10 mM TrisHCl, 0.01% NaN₃, pH 7.5 Injection volume: 20 μl Flow: 0.05 ml/min Runtime: 60 min and equillibration of additional 100 min

For this analysis the insulin derivatives of the invention are in a solution consisting of 0.6 mM derivative, 2.1 Zn²⁺/hexamer, 16 mM phenol, 7 mM phosphate pH 7.8. The retention time of the derivative is then compared to the retention times of the following standard molecules: Blue dextran (>5 MDa, K_(AV) 0.0), Thyroglobulin (669 kDa, K_(AV) 0.28), Ferritin (440 kDa, K_(AV) 0.39), Ovalbumin (44.5 kDa, K_(AV) 0.56), Ribonuclease (13.7 kDa, K_(AV) 0.69) and a second reference of Albumin (66 kDa, K_(AV) 0.53), Co(III)insulin-hexamer (35 kDa, K_(AV) 0.61), and monomeric insulin X2 (6 kDa, K_(AV) 0.73).

The following equation is used to determine the K_(av) for the derivative: K _(av)=(t−t ₀)/(V _(t)/(f+t _(d) −t ₀)) Where t is the retention time for a given peak, t₀ is the retention time for Blue dextran, V_(t) is the total column volume (here 2.4 ml), f is the flow (here 0.04 ml/min), and t_(d) is the retention time for Blue dextran without the column in the system.

The K_(av) value indicates the degree of selv-association of a derivative, i.e. a large K_(av) similar to the K_(av) for the Co(III)insulin hexamer and X2 insulin monomer shows low or no propensity of the derivative to form large, selv-associated complexes, while very small K_(av) close to zero or even negative shows great propensity of the derivative for selv-association into large, soluble complexes.

Hydrophobicity Data on Insulin Derivatives According to the Invention.

The hydrophobicity (hydrophobic index) of the insulin derivatives of the invention relative to human insulin, k′_(rel), was measured on a LiChrosorb RP18 (5 μm, 250×4 mm) HPLC column by isocratic elution at 40° C. using mixtures of A) 0.1 M sodium phosphate buffer, pH 7.3, containing 10% acetonitrile, and B) 50% acetonitrile in water as eluents. The elution was monitored by following the UV absorption of the eluate at 214 nm. Void time, t₀, was found by injecting 0.1 mM sodium nitrate. Retention time for human insulin, t_(human), was adjusted to at least 2t₀ by varying the ratio between the A and B solutions. k′_(rel)=(t_(derivative)−t₀)/(t_(human)−t₀). k′_(rel) found for a number of insulin derivatives according to the invention are given in Table 1.

Human Serum Albumin Affinity Assay

Relative binding constant of 125I-TyrA14-analogue to human serum albumin immobilised on Minileak particles and measured at 23° C. (detemir=1 in saline buffer).

Mixability of Long-Acting and Short-Acting Insulins as Analyzed by Size-Exclusion Chromatography of Insulin Mixtures

SEC: Mixability of Insulin Aspart (3 Zn/6 insulin, glycerol 1.6%, 16 mM phenol and 16 mM m-cresol, sodium chloride 10 mM, phosphate 7 mM, pH 7.4) and prolonged acting insulin (2.1 or 6 Zn/6 insulin) 30:70, as measured by collecting fractions from SEC (as described above) and quantifying by HPLC the presence of prolonged-acting and fast-acting insulins in the high molecular weight fraction (fraction 2, MW>HSA) and in the low molecular weight fraction (fraction 3, MW=HSA), respectively.

Four fractions are collected at size of 16 min after delay, of which fraction 2 [16-32 min] (peak 1) contain associated form larger than albumin (32 min correspond to K_(AV) 0.46) and fraction 3 (peak 2) contain dihexameric, hexameric, dimeric and monomeric forms of insulin.

HPLC: Reverse phase chromatography on a Zorbax Eclipse XDB-C18 2.1*15 mm (1.8 μm) gradient eluted with buffer A: 0.2 M sodium sulphate, 0.04 M sodium phosphate, 10% acetonitrile, pH 7.2 and buffer B: 70% acetonitrile at 30° C., 19-34% B in 4.5 min. linear, sudden initial condition at 5 min., run time of 7 min., flow of 0.5 ml/min., injection volume of 14 μL and UV detection at 276 nm using Insulin Aspart reference of 609 μM for both analogues.

Human Insulin serum receptor albumin Self- Hydrophobicity affinity affinity association: relative to relative to relative to K_(av) human human insulin (% area Compound insulin insulin detemir of peak) Example 1 ++ ++ ++ +++ 1.366 37% 1.9 0.0  (87%) 0.74 (13%) Example 2 ++ ++ ++ +++ 1.933 35% 2.2 0.0  (94%) 0.75  (6%) Example 3 ++ ++ + +++ 1.095 30% 0.37 0.08 (76%) 0.74 (24%) Example 4 +++ +++ + ++ 0.716 52%  0.069 0.15 (77%) 0.74 (23%) Example 5 ++ ++ ++ +++ 1.75  30% 1.93 0.02 (93%) 0.76  (7%) Example 6 ++ ++ ++ +++ 1.617 33% 1.36 0.01 (90%) Example 7 ++ ++ +++ +++ 2.936 37% 2.62 0.01  (90%). Example 8 ++ ++ ++ n.a. 2.461 38% 1.95 Example 9 +++ ++ + +++ 0.738 58% 0.06 0.05 (90%) 0.75 (10%) Example 10 ++ ++ + ++ 1.803 43% 0.33 0.26 (48%) Example 11 +++ ++ n.a. n.a. 0.435 43% Example 12 +++ +++ + ++ 0.989 49% 0.25 0.17 (44%) 0.7  (56%) Example 13 +++ +++ + + 0.552 59% 0.03 0.69 (100%)  Example 14 ++ ++ n.a. n.a. 1.61  48% Example 15 ++ ++ + ++ 1.09  47% 0.14 0.28 (42%) 0.72 (58%) Example 16 ++ +++ ++ ++ 2.21  88% 1.9  0.3  (28%) 0.52 (72%) Example 17 + ++ +++ ++ 15.9   12% 2.97 0.12 (87%) 0.79 (13%) Example 18 ++ ++ +++ ++ 4.16  36% 5.83 0.33 (24%) 0.5  (76%) Example 19 ++ ++ +++ ++ 3.73  42% 5.27 0.33 (24%) 0.51 (76%) Table legend: Hydrophobicity relative to human insulin: k'rel < 1: +++, 1-10: ++, >10: + (HI = 1) Insulin receptor affinity relative to human insulin: <5%: +, 5-50%: ++, >50%: +++ Human serum albumin affinity relative to insulin detemir: <0.5: +, 0.5-2: ++, >2: +++ Self-association: K_(av) < 0.1: +++, K_(av) < 0.55: ++ and K_(av) ≧ 0.55: + K_(av) = 0.55 for human serum albumin, K_(av) = 0.63 for human insulin Co(III)hexamer, K_(av) = 0.72 for the monomeric insulin analogue X2. n.a. = not analyzed.

Example 21 Euglycaemic Glucose Clamp after s.c. Administration of Insulin Preparations to Pigs

Female pigs, 60-90 kg, fasted for 18 h. During the experiments the pigs are free to move in their pens. An insulin dose is administered s.c., depending of dose size often divided in two depots. Each pig is kept euglycaemic at its individual fasting glucose levels for up to 24 h by a variable rate intravenous infusion of a 20% glucose solution. The infusion is given through a catheter inserted in the jugular vein. Depending on changes in plasma glucose concentrations observed during frequent plasma glucose monitoring, the necessary adjustments of the glucose infusion are made empirically. Blood samples are collected in EDTA glass tubes every 15-30 min, plasma separated for glucose and insulin measurements. Glucose is determined within 1.5 min of blood sampling with an YSI (Yellow Springs Instruments) glucose analyser (glucose oxidase method). Mean glucose infusion rate (GIR) profiles and mean plasma insulin profiles are made for each insulin preparation. (FIGS. 2-4 show mean±SEM).

Pharmacological Methods

Assay (I)

Insulin Receptor Binding of the Insulin Derivatives of the Invention

The affinity of the insulin analogues of the invention for the human insulin receptor was determined by a SPA assay (Scintillation Proximity Assay) microtiterplate antibody capture assay. SPA-PVT antibody-binding beads, anti-mouse reagent (Amersham Biosciences, Cat No. PRNQ0017) were mixed with 25 ml of binding buffer (100 mM HEPES pH 7.8; 100 mM sodium chloride, 10 mM MgSO₄, 0.025% Tween-20). Reagent mix for a single Packard Optiplate (Packard No. 6005190) is composed of 2.4 μl of a 1:5000 diluted purified recombinant human insulin receptor-exon 11, an amount of a stock solution of A14 Tyr[¹²⁵I]-human insulin corresponding to 5000 cpm per 100 μl of reagent mix, 12 μl of a 1:1000 dilution of F12 antibody, 3 ml of SPA-beads and binding buffer to a total of 12 ml. A total of 100 μl was then added and a dilution series is made from appropriate samples. To the dilution series was then added 100 μl of reagent mix and the samples were incubated for 16 hours while gently shaken. The phases were the then separated by centrifugation for 1 min and the plates counted in a Topcounter. The binding data were fitted using the nonlinear regression algorithm in the GraphPad Prism 2.01 (GraphPad Software, San Diego, Calif.).

Assay (II)

Potency of the Insulin Derivatives of the Invention Relative to Human Insulin

Sprague Dawley male rats weighing 238-383 g on the experimental day are used for the clamp experiment. The rats has free access to feed under controlled ambient conditions and fast overnight (from 3 pm) prior to the clamp experiment.

Experimental Protocol

The rats are acclimatized in the animal facilities for at least 1 week prior to the surgical procedure. Approximately 1 week prior to the clamp experiment Tygon catheters are inserted under halothane anaesthesia into the jugular vein (for infusion) and the carotid artery (for blood sampling) and exteriorised and fixed on the back of the neck. The rats are given Streptocilin vet. (Boehringer Ingelheim; 0.15 ml/rat, i.m.) post-surgically and placed in an animal care unit (25° C.) during the recovery period. In order to obtain analgesia, Anorphin (0.06 mg/rat, s.c.) is administered during anaesthesia and Rimadyl (1.5 mg/kg, s.c.) is administered after full recovery from the anaesthesia (2-3 h) and again once daily for 2 days.

The clamp technique employed is adapted from (1). At 7 am on the experimental day overnight fasted (from 3 pm the previous day) rats are weighed and connected to the sampling syringes and infusion system (Harvard 22 Basic pumps, Harvard, and Perfectum Hypodermic glass syringe, Aldrich) and then placed into individual clamp cages where they rest for ca. 45 min before start of experiment. The rats are able to move freely on their usual bedding during the entire experiment and had free access to drinking water. After a 30 min basal period during which plasma glucose levels were measured at 10 min intervals, the insulin derivative to be tested and human insulin (one dose level per rat, n=6-7 per dose level) were infused (i.v.) at a constant rate for 300 min. Plasma glucose levels are measured at 10 min intervals throughout and infusion of 20% aqueous glucose is adjusted accordingly in order to maintain euglyceamia. Samples of re-suspended erythrocytes were pooled from each rat and returned in about ½ ml volumes via the carotid catheter.

On each experimental day, samples of the solutions of the individual insulin derivatives to be tested and the human insulin solution are taken before and at the end of the clamp experiments and the concentrations of the peptides were confirmed by HPLC. Plasma concentrations of rat insulin and C-peptide as well as of the insulin derivative to be tested and human insulin are measured at relevant time points before and at the end of the studies. Rats are killed at the end of experiment using a pentobarbital overdose.

Test Compounds and Doses:

Insulins to be tested are diluted from a stock solution containing 97 μM of the insulin derivative in 5 mM phosphate pH 7.7. The final concentration in the solution ready for use is 0.45 μM of the insulin derivative, 5 mM of phosphate, 100 mM of sodium chloride, 0.007% of polysorbate 20. The pH was 7.7 and the i.v. infusion rate was 15 and 20 pmol·min⁻¹·kg⁻¹.

A stock solution of human insulin that is used as reference compound was formulated in a similar medium and infused i.v. at 6, 15 or 30 pmol·min⁻¹·kg⁻¹.

Both stock solutions are stored at −20° C. and thawed overnight at 4° C. before use. The solutions are gently turned upside down several times 15 min before they are transferred to the infusion syringes.

Assay (III)

Determination in Pigs of T_(50%) of the Insulin Derivatives of the Invention

T_(50%) is the time when 50% of an injected amount of the A14 Tyr[¹²⁵I] labelled derivative of an insulin to be tested has disappeared from the injection site as measured with an external γ-counter.

The principles of laboratory animal care are followed, Specific pathogen-free LYYD, non-diabetic female pigs, cross-breed of Danish Landrace, Yorkshire and Duroc, are used (Holmenlund, Haarloev, Denmark) for pharmacokinetic and pharmacodynamic studies. The pigs are conscious, 4-5 months of age and weighing 70-95 kg. The animals fast overnight for 18 h before the experiment.

Formulated preparations of insulin derivatives labelled in TyrA¹⁴ with ¹²⁵I are injected sc. in pigs as previously described (Ribel, U., Jørgensen, K, Brange, J, and Henriksen, U. The pig as a model for subcutaneous insulin absorption in man. Serrano-Rios, M and Lefèbvre, P. J. 891-896.1985. Amsterdam; New York; Oxford, Elsevier Science Publishers. 1985 (Conference Proceeding)).

At the beginning of the experiments a dose of 60 nmol of the insulin derivative according to the invention (test compound) and a dose of 60 nmol of insulin detemir (both ¹²⁵I labelled in Tyr A14) are injected at two separate sites in the neck of each pig.

The disappearance of the radioactive label from the site of sc. injection is monitored using a modification of the traditional external gamma-counting method (Ribel, U. Subcutaneous absorption of insulin analogues. Berger, M. and Gries, F. A. 70-77 (1993). Stuttgart; New York, Georg Thime Verlag (Conference Proceeding)). With this modified method it is possible to measure continuously the disappearance of radioactivity from a subcutaneous depot for several days using cordless portable device (Scancys Laboratorieteknik, Værløse, DK-3500, Denmark). The measurements are performed at 1-min intervals, and the counted values are corrected for background activity. 

The invention claimed is:
 1. An insulin derivative having a formula

wherein Ins is a parent insulin moiety and Q₁-Q₂-X₁-Q₃-Z is a substituent and where the Ins is attached to the substituent via an amide bond between the α-amino group of the N-terminal amino acid residue of the B chain of Ins or an ε-amino group of a Lys residue present in the A or B chain of Ins and a CO group in Q₁ or Q₂ of the substituent; Q₁ is: an amino acid amide of an amino acid with a carboxylic acid in the side chain, or an amino acid with an uncharged side chain, which residue forms, with its α-carboxylic acid group, an amide group together with the α-amino group of the N-terminal amino acid residue of the B chain of Ins or together with the ε-amino group of a Lys residue present in the A or B chain of Ins, or a chain composed of two, three or four residues, where each residue is, independently, an α-carboxy amino acid amide with a carboxylic acid in the side chain, or an amino acid residue with an uncharged side chain, where each residue forms, with its a-carboxylic acid group, an amide group together with either (i) one of the remaining residues forming the chain or (ii) the α-amino group of the N-terminal amino acid residue of the B chain of Ins or to the ε-amino group of a Lys residue present in the A or B chain of Ins, or a bond; Q₂ is: —COCH(CONH₂)— —COCH₂N(CH₂CONH₂)— —COCH₂N(CH₂CONH₂)COCH₂N(CH₂CONH₂) —COCH₂CH₂N(CH₂CH₂CONH₂)— —COCH₂CH₂N(CH₂CH₂CONH₂)—COCH₂CH₂N(CH₂CH₂CONH₂)— —COCH₂N(CH₂CH₂CONH₂)— —COCH₂CH₂N(CH₂CONH₂)— —COCH₂OCH₂CONH— —CO—((CR⁵R⁶)₁₋₆—NH—CO)₁₋₄— or —CO—((CR⁵R⁶)₁₋₆—CO—NH)₁₋₄—, where R⁵ independently can be H, —CH₃, —(CH₂)₁₋₆CH₃ or —CONH₂ and R⁶ independently can be H, —CH₃, or —(CH₂)₁₋₆CH₃; provided that if an amine in Q₂ forms a bond with the rest of the substituent, the amine must be bound to the rest of the substituent via a carbonyl group; Q₃ is —(CH₂)_(m)— where m is an integer in the range of 6 to 32; a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH₂— groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 32; or a divalent hydrocarbon chain of the formula —(CH₂)_(s)C₆H₄(CH₂)_(w)— wherein s and w are integers or one of them is zero so that the sum of s and w is in the range of 6-30; X₁ can be —C═O or a bond; Z is: —COOH; —CO-Asp; —CO-Glu; —CO-Gly; —CO-Sar; —CH(COOH)₂; —N(CH₂COOH)₂; —SO₃H; —OSO₃H; —OPO3H₂, —PO₃H₂ or tetrazol-5-yl; and any Zn² complex thereof wherein the insulin derivative is mixable with a fast-acting insulin with no blunting.
 2. The insulin derivative according to claim 1, wherein Z is —COOH.
 3. The insulin derivative according to claim 1, wherein the parent insulin is an insulin analogue.
 4. The insulin derivative according to claim 3, wherein the parent insulin is selected from the group consisting of: desB30 human insulin, GlyA21 human insulin, GlyA21desB30 human insulin, GlyA21ArgB31ArgB32 human insulin, LysB3GluB29 human insulin, LysB28ProB29 human insulin and ThrB29LysB30 human insulin.
 5. A pharmaceutical composition for the treatment of diabetes in a patient in need of such treatment, comprising a therapeutically effective amount of an insulin derivative according to claim 1 together with a pharmaceutically acceptable carrier.
 6. A method for producing a pharmaceutical composition comprising adding up to about 10 zinc atoms per 6 molecules of insulin derivative to an insulin derivative of claim
 1. 7. A method of treating diabetes in a patient in need of such a treatment, comprising administering to the patient a therapeutically effective amount of an insulin derivative according to claim
 1. 8. The method according to claim 7 for pulmonary treatment of diabetes.
 9. A mixture of an insulin derivative according to claim 1 and a rapid acting insulin analogue selected from the group consisting of AspB28 human insulin; LysB28ProB29 human insulin and LysB3GluB29 human insulin.
 10. An insulin derivative, wherein the insulin derivative is selected from the group consisting of: N^(εB29)-ω-carboxy-pentadecanoyl-γ-L-glutamylamide desB30 human insulin, N^(εB29)-ω-carboxy-pentadecanoyl-γ-amino-butanoyl desB30 human insulin, N^(εB29)-ω-carboxy-tetradecanoyl-γ-L-glutamylamide desB30 human insulin, N^(εB29)-ω-carboxy-tridecanoyl-γ-L-glutamylamide desB30 human insulin, N^(εB29)-ω-carboxy-pentadecanoyl-β-alanyl desB30 human insulin, N^(εB29)-ω-carboxy-pentadecanoyl-β-L-aspartylamide desB30 human insulin, N^(εB29)-ω-carboxy-pentadecanoyl-ε-aminohexanoyl desB30 human insulin, N^(εB29)-ω-carboxy-pentadecanoyl-δ-aminopentanoyl desB30 human insulin, N^(εB29)-ω-carboxy-tridecanoyl-γ-amino-butanoyl desB30 human insulin, N^(εB29)-ω-carboxy-undecanoyl-γ-amino-butanoyl desB30 human insulin, and N^(εB29)-ω-carboxy-tetradecanoyl-γ-amino-butanoyl desB30 human insulin.
 11. The insulin derivative according to claim 1, wherein Z is —CH(COOH)₂.
 12. The insulin derivative according to claim 1, wherein Z is —N(CH₂COOH)₂.
 13. The insulin derivative according to claim 1, wherein Z is —SO₃H.
 14. The insulin derivative according to claim 1, wherein Z is —PO₃H₂. 